THE HUMAN ANTIBODY RESPONSE TO SIMULTANEOUS INJECTION OF SIX SPECIFIC POLYSACCHARIDES OF PNEUMOCOCCUS* By MICHAEL HEIDELBERGER, PuD., COLIN M. MacLEOD, M.D., ann MARIE M. Dr LAPI (From the Department of Medicine, College of Physicians and Surgeons, Columbia University, and the Department of Microbiology, New York University College of Medicine, New York) (Received for publication, June 16, 1948) Previous quantitative studies of the antibody content of the sera of human beings injected with immunizing (1) doses of the specific polysaccharides of pneumococcus have dealt with analyses of sera after injection of two or three (reference 2) and four (reference 3) specific polysaccharides at one time. Since it was feasible for practical purposes to combine into a single solution as many as six polysaccharides of the pneumococcal types most commonly responsible for pneumococcal pneumonias in man, exact knowledge of the antibody response to the injection of so many chemically similar antigens appeared desirable. Data obtained with two different combinations of six polysaccharides are given in the present report. EXPERIMENTAL Medical student volunteers, after a preliminary bleeding (subscript 0), were injected subcutaneously with 1 ml. of a solution containing about 0.07 mg. each of six type-specific polysaccharides of pneumococcus. Subjects 201 to 206 received the specific polysaccharides of Types I, II, IX, V, VII, VII pneumococcus, these being the types most common in pneumo- coccal pneumonia of adults, while Nos. 207 to 212 received Types I, IV, VI, XIV, XVIII, XIX, the types most common in the pneumococcal pneumonias of children. Both antigen solutions were prepared by E. R. Squibb and Sons. Subject J, a woman 63 years of age, re- ceived the first solution. Nine months after its injection, sample J, was taken, when the antibodies formed were probably no longer at peak levels (2). From the other subjects a bleeding (subscript 1) was taken 6 weeks to 2 months after the injections. Analyses on sera 202, and 210, could not be completed owing to accidental contamination, and additional samples 2023 and 210, were procured 9.5 and 5.5 months after injection, respectively. Analyses were carried out according to the method mentioned in reference 4 and described in detail in reference 2. Precautions against bacterial contamination were taken owing to the large number of successive analyses required and the long standing necessary for each analysis. Superscripts after values for antibody nitrogen indicate an unusually large number of absorp- tions for complete removal of the antibody in question. *The work reported in this communication was carried out in part under the Harkness Research Fund of the Presbyterian Hospital and was also supported through the Commission on Acute Respiratory Diseases, Army Epidemiological Board, Office of The Surgeon General, United States Army. 369 370 ANTIBODY RESPONSE TO SIX PNEUMOCOCCUS POLYSACCHARIDES DISCUSSION The analytical values for the type-specific antibody content of the sera of hu- man beings injected subcutaneously with a mixture of six type-specific polysac- charides of pneumococcus resemble those obtained in earlier quantitative micro- TABLE I Micrograms Antibody Nutrogen Per 4 Ml. Pre- and Post-Immunisation Sera of Subjects Injected with Specific Polysaccharides of Pneumococcus Types I, 11, HI, V, Vil, and VIIT, 0.05 to 0.1 Mg. Bock emtboty | CuI CVI SI Sil $l Sv svit | svi | sxIv 2019 19 V7 0 5 10*, 15 0 0 4,3 3 201; 53 31 64 29 21 49* 7 202. 27 24 0 3 4*,4 0 0 0,0 0 202; 35 (14)t 15¢ 6 202 40 | 3,3 21 | 1%,11 | 0,0 0 | 41 2036 39 30 0 1 4*,3 0 1 0, 0 11 203, 52 33 33 21 107 36¢ 398 2040 2 14 0 0 o*,0 0 0 0, 0 0 204, 16 3 6 2 4 11? 18 2050 46 0 0 1" 0 3 6 205, 62 15 21 11 1 308 8 2065 32 0 0 0* 0 2 3 206, 28 8 18 5 0 7 1 Jo 23 0 0 4" 0 0 0 hi 16 15 13 10,6 | 12*,16 | 0,8 {431 17 C III = C-substance derived from Type III pneumococcus, C VI, from Type VI; S1,5S II, etc., refer to type-specific polysaccharides. & After removal of anti-C with C-substance derived from Type ITI and possibly containing traces of STII. ¢ Last analysis before contamination noted. analytical studies of the sera of subjects injected with two or three or four of the antigens at a time (2, 3). The total antibody response and the production of antibody to each antigen injected appeared to be as satisfactory as in the sub- jects treated with smaller numbers of polysaccharides. Variability of response and unequal reactivity of an individual to all of the type-specific polysaccha- rides in the mixture were encountered as frequently as in the previous series. In marked contrast to the behavior of the groups previously studied, roughly M. HEIDELBERGER, C, M. MACLEOD, AND M. M. Dr LAPI 371 one-half of the subjects showed increases in the anti-C content of their sera after the injections. Since almost all of the specific polysaccharides contain C-sub- stance as impurity, it is possible that the intensity of the antigenic stimulus for production of additional amounts of anti-C increases with the number of poly- saccharides used, and, hence, the probably greater quantity of C-substance injected. Whether or not all of this or only a portion is antigenic in man is not known. TABLE IIT Miécrograms Antibody Nitrogen Per 4 ml. Sera of Subjects Injected with Specific Polysaccharides of Pneumococous Types I, IV, VI, XIV, XVIII, and XIX, 0.05 te 0.1 Mg. Each antibody | oc SI si |} siVv svi | sxiv | sxvm | sSxIx 2070 27 1] 0 0 0 0 3 0 207; 26 54 9 2 47 9 6 208 6 0 0 0 0 0 5 0 2081 555 24 164 4 0 52 0 209» 31 0 0 2 1 4 5 1 20, 89 24 48 18 56 41 15* 2105 87 0 i Q 0 7 16 0 210; 158 50 23 210; 199 26 21 538 23 35 444 21lo 29 0 1 0 0 4 0 0 211; 32 5 § 1 9 5 0 2125 56 4 3 1 1 6 2 0 212, 57 19 5 8 70 9 14 * S ITI was not in the mixture injected. Additional data on normals were sought. Attention is also directed to the relatively large proportion of the subjects whose pre-immunization sera contained measurable quantities of antibodies ‘which precipitate with the specific polysaccharides of Types XIV and XVIII pneumococcus. SUMMARY 1, The antibody response in human beings after the injection of six type- specific polysaccharides of pneumococcus appears to be roughly of the same magnitude for each type as after the injection of fewer antigens. 2. A larger proportion of increases in anti-C was noted in the present series. 372 ANTIBODY RESPONSE TO SIX PNEUMOCOCCUS POLYSACCHARIDES 3. Many of the pre-immunization sera tested contained antibodies reactive. with the specific polysaccharides of Types XIV and XVIII pneumococcus. BIBLIOGRAPHY 1. MacLeod, C. M., Hodges, R. G., Heidelberger, M., and Bernhard, W. G., J. Exp Med., 1945, 82, 445. 2. Heidelberger, M., MacLeod, C. M., Kaiser, §. J., and Robinson, B., J. Exp. Med., 1946, 83, 303. 3. Heidelberger, M., MacLeod, C. M., Hodges, R. G., Bernhard, W. G., and Di Lapi, M. M., J. Exp. Med., 1947, 85, 227. 4, Heidelberger, M., and MacPherson, C. F. C., Science, 1943, 97, 405; 98, 63.