Studies on immunization against plague. I. The isolation and characterization of the soluble antigen of Pasteurella pestis. (1952)

Discussion and Summary Since the time of Markl (23) it has been recognized that immunogenic, toxic extracts of plague bacilli may be prepared. Rowland (3–6) found a soluble, toxic antigen capable of inducing immunity in rats and a water-insoluble portion devoid of immunogenic activity in rats. Schutze (13, 14) demonstrated, by means of serological tests, an antigen soluble at 56 C and destroyed by boiling, which he termed the “envelope” antigen, and an antigen stable and soluble at 100 C, termed the “somatic” antigen. Recently Seal (10) reported the isolation of various fractions of the plague bacillus. He used both sodium and ammonium sulfates and obtained substances precipitated by one third or one half saturation with these salts. They were all immunologically active but were not sufficiently well characterized to permit comparison with the fractions described in the present paper. In view of the properties of Fractions IA and IB, Seal9s fractions are probably mixtures of these two substances together with other material. In another paper Seal (11) reported the isolation of a serologically active polysaccharide. This substance was not characterized adequately. In the present investigation no evidence for the presence of such a polysaccharide was obtained. It may be that Seal9s polysaccharide is the carbohydrate moiety of Fraction IA. On the other hand, the data presented do not rule out the possibility that he had isolated a small amount of Fraction IA. Only further study can settle this question. The “envelope” protein described by Amies (12) may be Fraction IB. However, further work will be necessary to establish this identiy. Jawetz and Meyer (18), after a study of avirulent P. pestis strains, proposed that an antigenically complete strain contains an envelope antigen, a somatic antigen, a virulence factor (vi) and an antigen (avi) responsible for the immunogenic efficiency of some strains. They suggested that the only difference among all types of plague bacilli lies in quantitative differences in these hypothetical antigens. In the present study, some of these antigens were isolated, purified and characterized. Two proteins were isolated from the water-soluble fraction of P. pestis . These two proteins are nontoxic and have a high immunogenic value for mice and rats but are relatively poor antigens when tested on guinea pigs. They exhibit chemical and physical differences in that Fraction IA contains a carbohydrate moiety and is viscous in solution, while Fraction IB contains no carbohydrate, is relatively nonviscous, and can be crystallized from an ammonium sulfate solution. The two fractions are pure according to the criteria used. Fraction IB contains no carbohydrate and hence is not contaminated with Fraction IA. However, there is no certainty whether small amounts of Fraction IB are present in Fraction IA since their electrophoretic mobilities are essentially the same and adequate tests were not available to detect the presence of Fraction IB in Fraction IA. It is believed that both fractions are essentially free of other components of plague bacilli and of the culture medium. However, final proof of purity must await further evidence such as solubility determinations and ultracentrifugation studies. The biologic significance of the two fractions is not clear. Their electrophoretic and immunologic similarity suggest that the proteins of these fractions are identical and that the carbohydrate moiety of Fraction IA contributes neither to its electrophoretic properties nor to its immunologic specificity. It may be surmised that these two antigens are on the surface of the cell because antisera prepared against them agglutinate whole bacilli. The partial removal of these antigens from a suspension of plague bacilli results in a decrease in agglutination titer with such sera, but not with antisera prepared against whole bacilli. Since cultures of antigenically complete plague bacilli produce viscous colonies on suitable media, it may be that the surface antigen of intact plague bacilli is the viscous, carbohydrate-containing Fraction IA and that crystalline Fraction IB is formed from Fraction IB during extraction, through loss of the carbohydrate moiety. Attempts to separate the carbohydrate and protein portions of Fraction IA, however, were unsuccessful. In addition to Fractions IA and IB, the water-soluble portion of P. pestis contains a toxic substance which has been separated and concentrated, but neither purified nor characterized. Plague toxin will be the subject of a future communication. The water-insoluble residue contains antigenic substances of value for the guinea pig. This component of P. pestis has not been studied in detail, but some fraction of it is undoubtedly the “somatic” antigen of Schutze (13, 14). Further study may reveal several antigenic substances. For example in the present study no evidence has been found for the existence of a definite entity responsible for virulence, as Jawetz and Meyer (18) suggested. By implication, Otten (24) postulated the presence of an antigen to account for the fact that some avirulent strains induced good immunity in rats, but only a poor one in guinea pigs, while for other strains the reverse is true. These observations were confirmed by Jawetz and Meyer who used mice and guinea pigs. Whether varying quantities of the antigens described above are responsible for these differences is not certain. The strain differences observed by Otten might be due to quantitative antigenic differences. However, they may also be accounted for by differences in the ability of a given strain of P. pestis to grow and persist in a given species. Further investigations may reveal the explanation for the observations of Otten and of Jawetz and Meyer.