Pertussis antigens--screening models on toxicity.

From the same batch of B. pertussis bacteria two types of DPT-vaccines were produced after harvest of the inactivated organisms by centrifugation or acid precipitation. The first vaccine contained whole pertussis bacteria and the second an extracted antigen complex. In a screening programme with reliable animal tests, such as a modified mouse-weight-gain assay, mouse leukocytosis test, histamine sensitization in mice, allergic encephalitis in rats, and the limulus test it could be demonstrated that the vaccine with whole inactivated bacteria was more toxic than that with extracted antigens. Cell material harvested by centrifugation showed a lower rate of toxicity than that containing acid-precipitated pertussis organisms.

Serotype determinant protein of Neisseria Meningitidis. Large scale preparation by direct detergent treatment of the bacterial cells.

Neisseria meningitidis Group B microorganisms, inactivated with phenol and harvested by centrifugation, were subjected to direct treatment with various detergents to solubilize the serotype determinant proteins localized in the outer membrane. Analysis of the data showed that extraction of the cells with detergents provided yields of the serotype protein substantially exceeding those obtained by simple salt extraction of the bacteria. Routinely, more than 2 mg of end product per g of cell mass (wet weight) may be recovered by the present method. By gel chromatographic analysis, the serotype determinant protein was shown to interact with the capsular polysaccharides derived from Group A or C Neisseria meningitidis microorganisms, forming high molecular weight complexes. This interaction markedly enhanced the solubility of the serotype determinant protein. Combined vaccines of the type-specific protein with the group- specific polysaccharides were evaluated for their immunogenic potential in the subcutaneous steel spring implant model. In guinea pigs, amounts corresponding to 10 micrograms completely prevented infection upon challenge with homologous organisms four weeks after immunization. Partial protection was observed with immunizing doses corresponding to 2 micrograms or 0.4 micrograms/animal, respectively. Compared to lyophilized preparations, vaccines adsorbed to a mineral carrier were slightly less effective in inducing protection, whereas inclusion of Bordetella pertussis as a component of the vaccine stimulated the immune response.

Extracted protective antigen of Bordetella pertussis. I. Preparation and properties of the solubilized surface of components.

Bordetella pertussis microorganisms were treated with several extracting agents followed by ultracentrifugation to remove particulate matter. Analysis of the resulting supernatants by SDS gel electrophoresis showed one major component after simple salt extraction, and much more complex, although consistent pattern following detergent treatment. The yield of the solubilized protein in detergent extracts exceeded by far the values recorded for salt extracts. In order to prevent irreversible precipitation of the solubilized proteins upon removal of the denaturing agent, a novel procedure was developed. After extraction with urea-salt, the solubilized material was absorbed on a mineral carrier prior to the separation of the denaturing agent. The resulting absorbed vaccine was highly potent in the mouse-protection test, whereas the toxic reactions, elicited upon injection into experimental animals, were reduced in the comparison to the starting material. This diminished reactogenic potential was accompanied by the partial loss of the leukocytosis-promiting factor, whose activity was greatly diminished by urea-salt at alkaline pH-values. The procedure described may be applied to large-scale processing of Bordetella persussis microorganisms. Clinical trials now in progress should confirm or rebut the thesis that increased tolerability of the product, inferred from animal experiments, is reflected by fewer adverse reactions in humans. In the former case, the detergent extract vaccine may constitute a realistic alternative to conventional whole-cell vaccines against whooping-cough.