W. Brehmer

Biologically active components from mycobacterial cell walls: IV. protection of mice against aerosol infection with virulent Mycobacterium tuberculosis

Abstract Cell walls prepared from Bacillus Calmette-Guerin (BCG) and associated with oil droplets have been shown to protect mice from aerosol infection with the virulent H37Rv strain of Mycobacterium tuberculosis. A combination of the peptidoglycolipid cell wall skeleton (CWS-I), prepared by exhaustive organic solvent extraction of BCG cell walls, and P3, isolated by centrifugal microparticulate bed chromatography from the free lipids of the mycobacterial cell wall, gave protection comparable to that provided by whole BCG cell wall. Neither component by itself was capable of protecting mice. The mechanism of protection based upon the chemistry of the mycobacterial components is discussed.

IMMUNOTHERAPY WITH NONVIABLE MICROBIAL COMPONENTS

Structural components of microorganisms have been studied for immunopotentiating effect with the aid of transplantable (line 10) tumors in syngeneic guinea pigs. Microbial components were associated with oil droplets, suspended in Tween-saline, and injected intralesionally. BCG cell walls, given in this way, produced regression and cure of 50-60% of established tumors, as did viable BCG. Lipid extraction markedly reduced the tumor-regressing potency of cell walls, but P3, a trehalose mycolate present in the extract, restored full activity to the cell wall residue. P3 alone was nonsensitizing and had no antitumor activity, but it enhanced the latter property of various other microbial products. For example, the cure rates produced by cell walls of M. tuberculosis, M. bovis, M. phlei, or M. smegmatis were enhanced from 20-60% to as much as 90% by addition of P3. P3 also conferred antitumor activity on products from unrelated microbes, such as cell walls of E. coli, and in combination with endotoxins from rough Re mutant salmonellae, it produced cure rates of up to 93%. These results suggest that P3 is essential to the immunopotentiating activity of mycobacteria and that it may be broadly applicable in immunotherapy of cancer with microbial agents.

STRUCTURE AND BIOLOGICAL FUNCTIONS OF MYCOBACTERIA

The relationship between cell structure and function is obviously too large a subject to be covered in a short presentation. However, certain fundamental concepts can be considered by limiting ourselves to only one function of mycobacterial cells, that of their ability to stimulate an immune response in a suitable animal host. To investigate the relationship between acquired immunity in the host and the part of the whole bacterial cell which incites this immunity, it is necessary to accomplish two things: first, devise a suitable test system for immunity and, second, isolate the specific component of the mycobacterial cell which incites the immunity. To begin with, we must define what we mean by an immune response and, if possible, describe i t in a quantitative fashion. In our laboratory, we place greatest reliance upon a test system which involves challenge of mice by aerosol with small numbers of virulent tubercle bacilli and modification of the effects of such challenge by prior i.v. injection of experimental vaccines.13 Assessment of the response to vaccination includes: 1. The count of grossly visible lung lesions four weeks after challenge. 2. Enumeration of the bacilli in the target organ, the lung, at prescribed inter-

Immunization of mice by combinations of inactive fractions of Mycobacterium bovis strain BCG.

Summary In an attempt to identify the mycobacterial cell wall components which enhance resistance in mice to airborne infection with Mycobacterium tuberculosis H37Rv, 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 may be required in an effective vaccine.

Specificity of resistance to tuberculosis and to salmonellosis stimulated in mice by oil-treated cell walls.

Summary Coating with oil, which was essential to render cell walls of BOG protective to mice against challenge with tubercle bacilli by aerosol, has been found not to affect the specificity of reactions conditioned by cell walls in this and other systems. When the interval between vaccination and challenge was short (24 hours), mice were protected against heavy intravenous challenge with M. tuberculosis principally by nonspecific factors; after a 30-day interval, protection appeared to depend upon a mixture of specific and nonspecific effects.

Specific and nonspecific stimulation of resistance in mice against infection with mycobacterium tuberculosis H37Rv

Intravenous vaccination of mice with oil-treated cell walls of BCG orM. tuberculosis, H37Rv protected against aerosol challenge with virulent tubercle bacilli, whereas protoplasm of these strains or cell walls in aqueous suspension failed to elicit resistance.The aerosol challenge system has been shown to be the only test system for resistance against tuberculosis with a remarkable specificity, since cell walls of unrelated species likeS. typhimurium orL. monocytogenes were non-protective in this test, in contrast to the conventional “massive intravenous challenge test” where specific and non-specific factors can become effective.