Sonic extracts of B. pertussis were fractionated by chromatography employing DEAE-cellulose. Mouse protective antigen (MPA) was absorbed to the column and was found to be eluted over a range of salt concentrations from 0.05 to 0.35 M NaCl in dilute phosphate buffer, pH 7.2. The MPA was freed from nucleic acid impurities since these remained fixed to the column and could only be eluted with salt concentrations of 0.5 to 2.0 molarity. Some impurities of protein nature passed directly through the column. The measurable precipitating antigens associated with the crude extract were fractionated into two groups by the DEAE-cellulose. Dilute buffer elutions removed material that developed several cathodic bands by immunoelectrophoresis. More concentrated salt buffers eluted materials that exhibited one anodic band by immunoelectrophoresis against the whole B. pertussis antiserum. The measurement of significant potencies of MPA in the absence of either cathodic or anodic precipitinogens suggests that the latter represent impurities which may or may not have a relation to other known biological activities associated with B. pertussis (e.g., toxin).Certain discrepancies between mouse protective antigen and histamine-sensitizing factor based on the lack of proportionality of the activities from fraction to fraction suggest that their chemical natures are dissimilar.

The density gradient centrifugation of the histamine-sensitizing factor (HSF) of B. pertussis extracts in admixture with purified foreign proteins has revealed a property of the HSF not previously recognized. The HSF sedimentation was markedly accelerated in the presence of human serum macroglobulin and hog thyroglobulin. The absence of gross detectable changes in the protein profiles after the displacement of the HSF by sedimentation suggests the need for studies on the chemical composition of the HSF.The affinity of the HSF towards the foreign proteins, particularly those of large molecular size, has suggested a useful model by which the relationship of the HSF to other biological activities of B. pertussis may be studied.

In mice which were immunized with various immunizing agents 6 and 20 days prior to challenge, it was found that stimulation of lung clearance of intratracheally administered radioiodinated pertussis vaccine was observed chiefly in those mice which had received vaccine prepared from any of the three members of the genus Bordetella. The most marked effect occurred in the pertussis-immunized animals. In pertussis-immunized mice which were challenged intratracheally with heterologous radioiodinated antigens, the most significant increases in clearance were obtained in those animals receiving labeled Bordetella vaccines. When mice immunized with the various antigens were challenged by their labeled homologous antigens, again the most significant clearance stimulations occurred in the mice immunized and challenged with the Bordetella preparations. Clearance studies with mice immunized and/or challenged with a vaccine prepared from phase IV pertussis organisms would support a view that somatic antigens may stimulate lung clearance in mice immunized 6 days prior to challenge whereas the surface antigens may provoke a slower-developing and longer-lasting immunity that is responsible for the faster clearance observed 20 days after immunization.

The present study was undertaken to determine the cause of the wide variation in the response of mice following intranasal challenge with virulent H. pertussis. Mice, both normal and immunized, were inoculated intranasally with killed H. pertussis organisms labelled with iodine-131. The course of the tagged antigens, in the lungs and other organs of these animals, was followed by radioisotope counting. The lung uptake varied from 31.6% to 77.5% of the challenge dose in immunized animals and from 25.0% to 83.7% in non-immunized animals. It was considered that this variation was large enough to contribute substantially to the wide discrepancies found in animal assays. To date the cultures employed for intranasal challenge have all been of low virulence when administered by this route. A considerably more virulent strain of challenge organism than those in use now would be necessary to overcome the variations in the assay caused by uneven lung uptake of challenge culture. It was also found that the lungs of immunized mice were cleared more rapidly than the lungs of non-immunized mice in the 24-hr. period following the intranasal inoculation.

The intratracheal route of administration has been shown by means of radio-iodinated pertussis vaccine to give more reproducible deliveries to the lung tissue in the challenge of mice than the intranasal route. Mice immunized 1 day or longer with a single immunizing dose of pertussis vaccine cleared a significantly higher amount of radioactivity from their lungs for a period of 24 hours following the intratracheal administration of radioiodinated pertussis vaccine than did non-immunized mice. Among those animals that received the labeled vaccine intratracheally, the mice that were immunized 6 days to 30 days previously showed higher fresh weights of lungs than those not immunized. The significance of these results is discussed.