Isolation of a soluble component of Plasmodium berghei which induces immunity in rats

Soluble material was obtained from sonically freed plasmodiae by three procedures. Two procedures, cryo-impacting and freeze-thawing, were evaluated for their ability to disrupt the parasites and release soluble material. The soluble materials obtained by these procedures were compared to materials washed from the surfaces of sonically freed parasites. Between 35 and 40% of the total parasite protein was solubilized by freeze-thawing or cryo-impacting. One cycle of freeze-thawing released nearly as much protein as could be released by this method, and additional cycles of freeze-thawing had little additional effect. Cryo-impacting solubilized only a small amount of protein in addition to that which was released by the cycle of freeze-thawing inherent in the procedure. Reductions in the packed cell volume of the material remaining after freeze-thawing or cryo-impacting indicate that the insoluble fragments are broken into smaller pieces as treatment is extended. Electron microscopy of 30-s cryo-impacted and three-times freeze-thawed parasites revealed membrane fragments similar in appearance. Patterns obtained by polyacrylamide gel electrophoresis of the soluble material from freeze-thawed and cryo-impacted parasites were also similar, and approximately 13 protein bands were demonstrated. The material washed from the surfaces of the free parasites, on the other hand, resolved into only two to four major bands on the gel columns. In immunization studies, the soluble and insoluble fractions obtained by freeze-thawing or cryo-impacting and the material washed from the surfaces of the parasites all stimulated a protective immune response. On the basis of the amount of protein required to stimulate roughly comparable immunity, the soluble fraction obtained by freeze-thawing or cryo-impacting free parasites was about twice as potent an immunogen as was the insoluble fraction. The material obtained by gentle washing of the freed parasites was approximately 20 times as potent an immunogen as were the freed parasites and about 7 times as potent as the soluble material obtained by freeze-thawing or cryo-impacting.