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Ribi, E. (Rocky Mountain Laboratory, Hamilton, Mont.), R. L. Anacker, W. Brehmer, G. Goode, C. L. Larson, R. H. List, K. C. Milner, and W. C. Wicht. Factors influencing protection against experimental tuberculosis in mice by heat-stable cell wall vaccines. J. Bacteriol. 92:869–879. 1966.—Studies of nonviable, heat-stable vaccines for active protection against experimental tuberculosis have been continued with a test involving aerosol challenge of intravenously vaccinated mice. The previously reported activating effect of light mineral oil on disrupted cells of the BCG strain was found to be shared by certain other mineral oils and a synthetic, 24-carbon hydrocarbon, but not by kerosene or any of several vegetable oils. Dry cell walls coated with a small amount of oil and dispersed in saline with aid of an emulsifier were suitable for intravenous administration and were effective in promoting resistance to challenge. Oil used in this manner, in contrast to water-in-oil emulsions of the Freund type which could not be administered intravenously, did not potentiate the tuberculin-sensitizing activity of the cell walls. Although the amount of oil required for full effect was small (< 0.5 ml/100 mg of dry antigen), there was a critical level below which optimal enhancement was not achieved. More stable suspensions than could be obtained with the other oils were readily prepared from cell walls treated with the synthetic hydrocarbon, 7-n-hexyloctadecane. Extended experience has shown that in this test system both the viable BCG standard vaccine and heated, oil-treated experimental vaccines gave highly reproducible results showing graded responses to graded doses.

Anacker, R. L. (Rocky Mountain Laboratory, Hamilton, Mont.), W. D. Bickel, W. T. Haskins, K. C. Milner, E. Ribi, and J. A. Rudbach. Frequency of occurrence of native hapten among enterobacterial species. J. Bacteriol. 91:1427–1433. 1966.—Smooth cultures of representative Enterobacteriaceaewere screened for the presence of native hapten, a substance previously extracted with trichloroacetic acid from the protoplasmic fraction of one strain each of Escherichia coliO111:B4 and O113. Trichloroacetic acid extracts of protoplasmic fractions of the cells were analyzed for chemical composition, for constituent sugars by paper chromatography, for immunochemical relationship to endotoxin purified by gel filtration, for sedimentation behavior, and for pyrogenicity in rabbits and lethal toxicity in chick embryos. Extracts from two of three additional strains of E. coliO113, all five additional strains of E. coliO111:B4, and one strain each of E. coliO26:B6 and O55:B5 were similar to previously described native hapten in chemical composition, sedimentation properties (S20,w, 3.7 to 5.2), biological potency (usually less than 0.1% that of corresponding endotoxin), and immunochemical relationship to endotoxin. Extracts of one strain each of E. coliO127:B8, Serratia marcescens, Citrobacter freundii, Salmonella enteritidis, and of two lipopolysaccharide-deficient mutants of S. enteritidisdiffered from typical native hapten. The biosynthetic relationship of native hapten to endotoxin has not yet been revealed.

Anacker, R. L. (National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratory, Hamilton, Mont.), R. A. Finkelstein, W. T. Haskins, M. Landy, K. C. Milner, E. Ribi, and P. W. Stashak. Origin and properties of naturally occurring hapten from Escherichia coli. J. Bacteriol. 88:1705–1720. 1964.—Haptens found in preparations of endotoxin and in fractions of disrupted cells, particularly one termed “native hapten,” which appeared to be associated with the protoplasm of cells rather than with cell walls, have been further investigated with a view to establishing their origin and composition as well as their host-reactive properties. For this purpose, cells from a smooth strain of Escherichia coliO111:B4 were either extracted directly or disrupted and separated into cell-wall and protoplasmic fractions. Haptens were obtained by gel filtration of endotoxins, by trichloroacetic acid extraction of protoplasm, and by a mild acid hydrolysis of endotoxin. Several lines of evidence indicated that native hapten originated in the protoplasm rather than by autolysis or degradation of cell-wall endotoxin during procedures employed in disruption. In gel diffusion and quantitative precipitin tests, no hapten was identical with endotoxin, but native hapten was serologically the most complex of the haptens and precipitated the most antibody. Native and acid haptens, on a weight basis, fixed about 1% of the quantity of complement fixed by homologous endotoxin. Haptens did not stimulate the production of antibodies in mice or rabbits and did not elicit endotoxic host reactions. Chemically, native hapten differed from endotoxin and from acid hapten in that it lacked phosphorus, heptose, long-chain fatty acids, and 2-keto-3-deoxyoctonate. These substances did not appear to be determinants of antigenic specificity, but they may provide necessary bonds for assembling hapten-like units into fully antigenic and toxic macromolecules.

Anacker, R. L. (Rocky Mountain Laboratory, Hamilton, Mont.), W. T. Haskins, D. B. Lackman, E. Ribi, and E. G. Pickens. Conversion of the phase I antigen of Coxiella burnetiito hapten by phenol treatment. J. Bacteriol. 85:1165–1170. 1963.—Trichloroacetic acid extracts of Coxiella burnetiiare converted to hapten by treatment with phenol. Such extracts react, like the original trichloroacetic acid extract, at high dilution in the complement-fixation test and produce zones of precipitate with specific antibody in gel diffusion tests; but, unlike the parent extract, injection of the phenol-treated extract neither induces resistance to challenge in guinea pigs nor antibody formation in guinea pigs, rabbits, or mice. This loss of antigenicity is correlated with removal of protein from the original product.

In an attempt to identify the mycobacterial cell wall components which enhance resistance in mice to airborne infection with Mycobacterium tuberculosisH37Rv, fractions of BCG were prepared by organic solvent extraction and by alkali or lipase treatment. Although certain of these fractions, when tested individually, were impotent in our protection test, vaccines containing combinations of solvent-extracted, alkali-treated, or lipase-treated cell walls and of an inactive chloroform or methyl alcohol extract were significantly effective. The wax D fraction, but not the wax B or C fractions, of the chloroform extract in combination with “inactive” cell walls was highly protective. Since combinations of either the “inactive” BCG cell wall residue or the BCG chloroform extract with heterologous substances failed to enhance resistance of mice, two or more specific and distinct mycobacterial components maybe required in an effective vaccine.

Minute amounts of aqueousether extracted S. enteritidisendotoxin, upon intravenous injection into rabbits, alter dermal reactivity to intradermally injected epinephrine. The lipid A component is only approximately 1/2000 as active as the endotoxin when potent endotoxins are tested. Phenol-water treatment of aqueous-ether extracted endotoxin reduces this activity.