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It has repeatedly been observed that various bacterial preparations could increase the host's resistance to tumors. It has also been shown that after nonspecific activation by BCG (bacillus Calmette-Guérin), peritoneal macrophages could inhibit in vitro the growth of neoplastic target cells. In the present study a fraction extracted from Myobacterium smegmatis and referred to as interphase material was tested in view of measuring its ability to activate macrophages in vitro and in vivo. This preparation was previously shown to protect mice against a syngeneic leukemia and to increase the immune response of the guinea pig. Other water-soluble adjuvants devoid of demonstrable antitumor activity in vivo were also assayed. The results argue in favor of a correlation between adjuvant activity and the capacity of activating macrophages. Moreover, interphase material administered in vivo consistently induced stronger and more persistent stimulations of macrophages than the other preparations assayed.

Two purified peptidoglycans extracted from E. coli or B. megaterium strongly stimulated the spleen lymphocytes of rabbits and of normal or nude mice. Both preparations can substitute for Mycobacteria in Freund's complete adjuvant. The peptidoglycan extracted from M. lysodeikticus by a similar procedure which lacks adjuvant activity, did not induce blast transformation. However, the monomere of the E. coli peptidoglycan was devoid of mitogenicity although it has also a marked adjuvant activity.

A water-soluble extract of Nocardia opaca (NWSE) induced a very strong stimulation of rabbit and mouse spleen lymphocytes and a weak, but significant, increase of thymidine incorporation by human peripheral lymphocytes and monkey blood, or spleen cells. As was demonstrated by the migration inhibition factor assay, the strong mitogenic activity observed with mice spleen cells was not related to a state of natural impregnation. In the same experiments, the various lymphocytes were also incubated with M. smegmatis water-soluble adjuvant (WSA) and S. enteritidis lipopolysaccharide (LPS). No mitogenic activity was shown with WSA even when this extract was incubated with the lymphocytes of human tuberculin responders, showing that this preparation is free of tuberculoprotein contaminants. Finally, it must be noted that, contrary to what is observed in mice, LPS did not induce thymidine incorporation by monkey or rabbit spleen cells.

Nocardia water soluble mitogen (NWSM) is known to stimulate mouse and rabbit lymphoid cells and to act selectively on murine B-derived lymphocytes. In this paper, evidence is presented that NWSM is also a mitogen for rabbit bursal equivalent cells and does not bring about blast transformation of thymus-derived rabbit cells. Pretreatment of rabbit spleen lymphocytes with antibody directed against rabbit thymus lymphocyte antigen (anti-RTLA serum) and with complement did not affect the strong increase of thymidine incorporation which follows stimulation with NWSM. The mitogen-induced polyclonal activation of antibody-forming cells and resulted in the presence of 30% of cells with the ultrastructural characteristics of plasmocytes. These observations led to the conclusion that NWSM is a mitogen for a B-derived lymphocyte of the rabbit.

Digestion by lysozyme of delipidated cells of Mycobacterium smegmatis liberates a water-soluble immunoadjuvant fraction which is chemically very similar to the water-soluble adjuvant (WSA) obtained previously from purified cell walls, but which contains somewhat more non-peptidoglycan amino acids. The yield of peptidoglycan-arabinogalactan complex is about 10 times greater starting from whole cells than from cell walls. The main biological properties of this “neo-WSA” are described: it increases circulating antibodies to ovalbumin in guinea pigs, it does not produce polyarthritis in rats or induce hypersensitivity to tuberculin, it does not increase susceptibility to histamine or hyperreactivity to endotoxin, and does not produce spleen and liver hypertrophy. Analogous immunostimulant fractions have also been obtained from delipidated cells of Nocardia opaca by lysozyme treatment.

Summary The bactericidal effect of normal mouse serum, generally ascribed to natural “O” antibodies, can be removed by absorption with rough microorganisms. Passive hemagglutination tests disclose the presence of thermolabile, non-agglutinating “R” antibodies in normal mouse serum; titers in germ-free animals are much lower. Horse hyperimmune serum against rough S. typhimurium protects mice against infection with unrelated virulent smooth K. pneumoniae. Provided the Klebsiellae are preincubated in normal mouse serum bactericidal activity can also be demonstrated in vitro. This activity does not require the presence of complement and can be removed either by the same Klebsiella strain or by rough strains of Salmonellae but not by the smooth strain of Salmonellae from which these mutants originate. Although these effects would most easily be accounted for by specific antibodies it is also possible that they are instead attributable to a serum protein capable of charge interaction with rough antigens. In any event these findings, together with previous observations concerning degradation of somatic antigen and phenotypic modification of Klebsiellae recovered from endotoxin-treated mice, have led to the development of a new concept of natural immunity to Enterobacteriaceae. In this hypothesis, enzymatic factors capable of attacking cell wall components unmask R antigenic sites common to many bacterial strains and species. Thereafter the presence of a few types of “R” antibodies or of serum factors reacting with rough antigens have the capability of coping, like masterkeys, with a very wide range of infections due to serologically unrelated organisms.

A streptomycin-resistant strain of K. pneumoniae obtained after mutation in vitro was found to be less virulent than the sensitive strain in mice. However, a single injection of lipopolysaccharides (LPS) administered 24 hours before challenge increased the host's resistance to both strains. In contrast, the virulence was not changed in K. pneumoniae accepting R-factors for ampicillin, streptomycin, chloramphenicol, tetracycline and sulphonamide. The plasmids were transferred to K. pneumoniae from two strains of Escherichia coli possessing different R-factors with the same resistance pattern. As in the first case, mice pretreated by endotoxin were protected against a challenge by microorganisms carrying R-factors. The capacity of the stimulated host to destory resistant or sensitive organisms was of the same order. Klebsiella recovered 5 or 24 hours after infection from the blood, liver and spleen did not lost their antibiotic-resistance. In this study, BCG and Corynebacterium granulosum were also used. Like LPS, these two immunostimulants protected very effectively mice infected with K. pneumoniae rendered resistant to antibiotics by R-factor transfer.

Though highly efficient, Freund's adjuvant has serious limitations because it contains mineral oil and isomannide monooleate emulsifier (arlacel A). The present study describes the conditions under which a stable water in oil emulsion can be produced by using metabolizable peanut oil with arlacel. When mycobacteria are added, a potent emulsified oil adjuvant is obtained which increases the immune response to BSA and to influenza vaccine.

McIntire and al. have observed that a lipopolysaccharide (LPS) extracted from E. coli could be detoxified by succinylation or phtalylation and remained capable of enhancing the immune respose to serum albumins. The data reported here confirm that several LPS preparations after treatment by phtalylation retained their adjuvant activity when injected with bovine serum albumin or influenza vaccine. Yet their toxicity (as measured in adrenalectomized mice) was at least 10 000-fold smaller. Furthermore it was observed that after phtalylation LPS could still induce blastic transformation of murine B-derived lymphocytes. Thus it was possible to dissociate the toxicity of LPS from both its adjuvant and mitogentic activities.

The use of mycobacteria in cancer therapy, either as systemic or as local immunoadjuvants, has been proposed [9, 11, 16]. These microorganisms can enhance the resistance of the host to a variety of attacks (bacterial, protozoal, viral or tumoral) by nonspecific mechanisms. Because they are powerful immunoadjuvants, they can also potentiate specifically the immune response to a given antigen. This capacity may be particularly valuable in the case of neoantigens that are weak immunogens. In recent years, both approaches have been used clinically and experimentally [11, 13, 20]. BCG (Mycobacterium tuberculosis, bacillus Calmette-Guerin), Hemophilus pertussis and Corynebacterium parvum are amongst the most popular candidates for clinical and animal experimentation in this field. H. pertussis and more recently C. parvum have been shown to increase the susceptibility of mice to histamine [3, 14] and may therefore expose the host to a wide variety of allergic and physiological accidents. Although BCG lacks this histamine-sensitizing activity, it is still capable of inducing many of the noxious effects observed after the injection of H. pertussis or C. parvum. Thus, killed BCG administered in saline sensitizes to tuberculin, produces lymphoid hyperplasia and increases the reactivity of mice to endotoxins [8, 18]. The decreased resistance towards lipopolysaccharides may be the cause of a great number of pathophysiological disturbances. Mycobacteria administered as a water-in-oil emulsion also produce allergic polyarthritis in the rat, a response which has been considered to indicate autoimmune disease [17]. Several investigators have suggested that insoluble Wax D extracted from virulent strains was the immunoadjuvant component of mycobacterial cells and that this substance is also responsible for allergic polyarthritis [17, 19].

Adam et al. [1] showed that a water-soluble fraction (WSA) extracted from mycobacteria was a potent adjuvant to various antigens when added to Freund’s incomplete adjuvant (FIA). Furthermore, this substance was free of the untoward effects of whole mycobacteria: WSA did not sensitize to tuberculin, did not increase the susceptibility of mice to endotoxin nor did it induce allergic polyarthritis in rats [4]. Under certain conditions, WSA in saline also potentiated the immune response [10].

It has long been recognized that certain microorganisms or bacterial extracts increase the host’s resistance to a variety of unrelated bacterial or viral infections and also stimulate immunity against tumors [4, 14, 16, 21, 24, 31, 18]. It seems reasonable to assume that evolutionary pressure has preserved feedback mechanisms in the host-parasite relationship, such as stimulation of the host by certain substances of the infective agent, e.g. the lipopolysaccharide of gram-negative bacteria [9]. However, when compared to endotoxins, which also enhance nonspecific immunity very strongly [8, 24] mycobacteria appear to possess 2 major advantages: 1. they can trigger cell-mediated immunity 1, which probably plays the major role in resistance, particularly to tumors; 2. some of the varied biological activities of mycobacteria can be related to different components which can be separated by chemical fractionation.