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In the preceding communications, the intracutaneous vaccination of rabbits with Pneumococcus (S forms) has been shown to give rise chiefly to the formation of the antiprotein rather than the type specific antibodies, and to the development of an increased resistance to infection with organisms of homologous and heterologous types. The present paper describes briefly the development of an altered tissue reaction to Pneumococcus and its protein derivatives in rabbits which have been inoculated repeatedly into the skin with heat killed suspensions of R and S pneumococci.Mackenzie and Woo1have shown that guinea pigs, injected intracutaneously with an alkaline extract of Pneumococcus, develop an allergic reaction in the skin to the bacterial protein; Zinsser and Grinnell2have produced allergic sensitization to pneumococcus autolysates in guinea pigs previously injected intradermally or intraperitoneally with the same material. Bull and McKee3have recently shown that rabbits, after recovery from infection induced by intranasal inoculation of pneumococci, are highly skin-sensitive to pneumococcus autolysate.The present observations were made in the course of a study of the antibody response and the immunity developed as a result of intracutaneous injection of rabbits. The intracutaneous injection in normal rabbits of 0.2 cc. of a heated vaccine, representing the bacteria from 2 cc. of broth culture, is followed by the appearance locally of a circumscribed slightly raised and indurated nodule, reddish in color, and measuring about 1 cm. in diameter. Upon repeated injection at weekly intervals the reaction changes in character; the size increases, often reaching a maximum of 4 to 6 cm. in diameter, accompanied by a spreading edema and purplish discoloration. The maximum reaction is generally reached after 6 to 8 injections and thereafter each successive lesion tends to, become less intense but to persist longer, often breaking down with the discharge of sterile necrotic material.

The fact that virulent, type-specific, capsulated, “S” forms of pneumococcus can be degraded to avirulent, non-type-specific, unencapsulated, “R” forms is well established. (Friel,1Stryker,2Griffith,3Amoss,4and Reimann.5) The reverse process, however, the conversion of avirulent, “R” forms into virulent, “S” forms, has been a matter of some controversy.Felton and Dougherty,6by means of an automatic transferring device in a milk-containing medium, were able to restore virulence to a strain of pneumococcus which had become completely avirulent. They employed pure line strains derived from single cells, and report that some cultures showed a maximal increase in virulence while others remained avirulent. Their investigation preceded the recognition of “R” and “S” forms of pneumococcus. Griffith3states that “R” strains may revert in all respects to the “S” type or remain unchanged after many generations in subculture or plain blood broth. Single cell cultures, however, were not employed. Reimann5reports that he was unable to enhance the virulence of an “R” strain derived from Type I even after 105 mouse passages. Levinthal7was able to effect the transformation by growing “R” forms in serum broth at 25° C. and subsequently passing the cultures through mice.For bacteria other than pneumococcus the same differences of opinion appear to exist. Recently, however, Jordan11has demonstrated the interconvertibility of “R” and “S” forms of B. paratyphosus B, and Soule12has shown the reversibility of the dissociated form of B. subtilis.

Suspensions of heat killed pneumococci were injected into the skin of rabbits at intervals of 7 days during a period of 10 to 14 weeks. The total amount of bacterial substance injected was equivalent to and often greater than that ordinarily employed in the routine immunization of rabbits by the intravenous method. Pneumococci of Types I and III and a degraded “R” strain derived from Type II were used. The sera of the treated rabbits were tested for the presence of agglutinins, precipitins, and protective antibodies. The serum obtained from 85% of the animals immunized intracutaneously with Type I pneumococcus failed to show the presence of any demonstrable type specific antibodies. Virulent cultures of Type I were not agglutinated, nor were solutions of the specific soluble substance from organisms of the homologous type precipitated by these sera even when used in high concentrations. Only rarely did the serum confer any passive protection upon mice infected with a virulent strain of Type I, and in these instances the protective titre was low. In only 15% of animals studied was there any serological evidence of type specific response to repeated intracutaneous inoculation of Type I organisms; in these instances the presence in the serum of specific agglutinins was demonstrable only in low dilutions varying from 1:1 to 1:20.In terms of its capacity to stimulate the formation of type specific antibodies, Type III pneumococcus is at best a poor antigen. It was to be expected, therefore, that organisms of this type, when injected intracutaneously into rabbits, would fail to elicit the type specific response. Such proved to be the case. None of the rabbits treated by the skin method developed demonstrable serum antibodies against Type III.

In the preceding communication it was shown that the antibody response elicited in rabbits vaccinated intracutaneously with specific type strains of Pneumococcus is characterized by the presence in the serum of antiprotein antibodies in excess, together with a corresponding decrease or complete absence of type specific antibodies. That this change in the order of specific antibodies is related to the mode of inoculation was shown by the fact that the same strain (Type I Pneumococcus), when injected intravenously, invariably stimulates the formation of the dominant type-specific antibodies.The present paper summarizes the results of a study of the occurrence and nature of the active resistance to infection which develops in rabbits vaccinated intracutaneously with R and S forms of Pneumococcus. Briefly, it may be stated that, following repeated skin inoculation of heat-killed suspensions of type-specific pneumococci or of the R variants, rabbits acquire a considerable degree of active immunity against infection with virulent strains of homologous and heterologous types. For example, rabbits, immunized intracutaneously with Type I may survive the intravenous injection of a dose as large as 0.2 cc. of homologous culture, the virulence of which is such that 0.0000001 cc. kills normal control animals. Similarly, rabbits immunized in the same manner to an avirulent strain of Type III may survive an infecting dose of from 0.5 to 1 cc. of a culture of the homologous type which by animal passage has been rendered so virulent that 0.001 cc. to 0.0001 cc. is sufficient to kill normal rabbits. Furthermore, animals inoculated intracutaneously with Type III become as resistant to subsequent infection with Type I as animals similarly immunized with homologous organisms. Even when an R strain of Pneumococcus derived from Type II is used for injection, rabbits acquire a high degree of resistance against infection with virulent Type I organisms.