Isolation of a proline-rich mycobacterial protein eliciting delayed-type hypersensitivity reactions only in guinea pigs immunized with living mycobacteria.

Effective protection against a virulent challenge with Mycobacterium tuberculosis is induced only by a previous immunization with living attenuated mycobacteria, usually bacillus Calmette-Guérin (BCG). Living and killed bacteria share a number of common antigens. To identify and to purify molecules that are dominant antigens during immunization with living bacteria, a two-step selection procedure was used. Quantitative delayed-type hypersensitivity (DTH) reactions elicited in guinea pigs immunized either with living or with killed BCG were used to select or counterselect antigens present in BCG culture filtrates. Each major fraction eluted from a series of HPLC columns (gel filtration, DEAE, reverse-phase chromatography) was assayed and titrated on guinea pigs of each group. A protein with an unusual amino acid composition (40% proline, 12% threonine) was purified and N-terminally sequenced. To our knowledge, the sequence Thr-Pro-Pro-Xaa-Glu-Xaa-Pro-Pro-Pro-Pro-Gln-Xaa-Val-Xaa-Leu has not been previously reported. The protein was 100-fold more potent on guinea pigs immunized with living bacteria than on guinea pigs immunized with dead bacteria to elicit a DTH reaction.

M. leprae and PPD-triggered T cell lines in tuberculoid and lepromatous leprosy.

Proliferative responses of peripheral blood mononuclear cells (PBMC) to Mycobacterium leprae and bacillus Calmette Guerin-derived purified protein derivative (PPD) were studied in the presence or absence of interleukin 2 (IL 2) in high M. leprae responders (tuberculoid leprosy patients and healthy subjects) and low M. leprae responders (lepromatous leprosy patients). High responders in most cases developed a strong proliferative response to both antigens in the absence of IL 2. Additional IL 2 and restimulation with antigen plus autologous antigen-presenting cells (APC) allowed the derivation of antigen-specific T cell lines. The lines were assayed for proliferative responses to several mycobacterial antigens. Both PPD and M. leprae-triggered T cell lines exhibited a good proliferative response to either antigen and showed in addition a broad cross-reactivity with other mycobacteria, suggesting a preferential T cell response to epitopes shared by several mycobacterial species. Within the lepromatous group, 50% of the patients studied could mount a proliferative response to PPD antigen in the absence of IL 2, but none of them was able to do so with M. leprae antigen. The addition of IL 2 increased the number of positive responders to PPD in this group, and in some patients IL 2 was able to restore M. leprae reactivity as well, suggesting that IL 2 had overcome a suppressor mechanism. PPD and M. leprae-triggered T cell lines were obtained from these subjects (with IL 2 added from the beginning of the culture when required). M. leprae lines exhibited variable and unstable pattern of specificity, most lines exhibiting, at least transiently, a cross-reactive response to other mycobacteria, but some displaying only M. leprae-specific response. In contrast, PPD lines from these subjects consistently exhibited a good response to PPD, a lesser response to various other mycobacteria and no response to M. leprae, a pattern differing from that obtained with PPD lines of high M. leprae responders. Co-cultures of irradiated lepromatous PPD triggered T cell lines with fresh autologous PBMC non-specifically reduced the proliferative response of the latter to PPD, as well as to unrelated antigens. A similar suppression was also observed when PPD lines from one of the tuberculoid patients were assayed. PPD and M. leprae T cell lines from both high and low responders initially exhibited the same CD4+ CD8- phenotype. In all cases, antigenic specificity declined and could not be maintained after 5 to 8 wk of continuous culture, a change associated with the progressive appearance of CD8+ and Leu8+ cells.

Apparent increased sensitivity of mice to tuberculin by adding a non-specific inflammatory agent to the antigen.

The delayed-type hypersensitivity (DTH) reaction at the site of tuberculin injection in immunised animals depends on the presence of sentisitised T-lymphocytes which interact with the antigen and recruit non-specific phagocytic cells. The intensity of DTH reaction was found to be related to the non-specific inflammatory stimulus created by antigen injection. The early plasma protein extravasation which occurred 0.5 of an hour after antigen injection was correlated with the intensity of DTH reaction measured 18 hours later. The addition to tuberculin of a non-specific inflammatory agent (concanavalin A or sheep red blood cells) in a dose range without clinical inflammatory effect increased the apparent potency of tuberculin by a factor 1000.

Search of a standard for the titration of antigen L.

The Antigen L is made of the 10 p.100 TCA precipitable proteins that remained after 2 p.100 TCA proteins precipitation of a live BCG culture filtrate. It is 6 times more active in guinea pigs sensitized by living BCG than by inactivated BCG. The reference tuberculin (BR6) is the fraction retained by DEAE-Cellulose chromatography of the purified TP tuberculin described elsewhere (2). The kinetic study of BR6 using guinea pigs sensitized by live or inactivated BCG showed that between the 2nd and 4th month, BR6 had the same activity in both cases in respect to the diameter of the erythema reactions. Therefore, BR6 may serve as reference for the Antigen L between the 2nd and 4th month of sensitization.

Evaluation of difficulties in the standardization of human and bovine tuberculins.

The tuberculins are made of mixtures of antigens. Certain elements are common to all tuberculins, others are particular to each one. Bovine and human tuberculins are widely different. This is the reason why the activities of a bovine tuberculin using a human tuberculin standard will be in a ratio of 1 to 5 according to whether the guinea-pigs were sensitized with human or bovine mycobacteria. Also, the activities measured for a given tuberculin might be different if the mycobacteria used in sensitization were inactivated or not, depending on the amount of Antigen L in the tuberculin or in the standard. Because BCG is well defined and used worldwide we propose that a new standard be prepared from BCG cultures, and that the animals be sensitized by live BCG. Two new units are proposed: Bovine Clinical Unit (B.C.U.) and Human Clinical Unit (H.C.U.).

Preparation of tuberculin antigen L.

The preparation of antigen L using culture filtrates of live BCG is described. The procedure consisted essentially of precipitating this filtrate with 10% trichloroacetic acid in the presence of N-butyl alcohol 4% final concentration. The precipitate was chromatographed on carboxy-methyl-trisacryl-M and then on DEAE-trisacryl-M; in both cases, maximal biological activity (delayed-type hypersensitivity) was found in the void volume. The product thus obtained was chromatographed on a molecular sieve so as to eliminate poorly active fractions. The final product was 30-fold more active on guinea-pigs sensitized with live BCG than on those sensitized with inactivated BCG.

[Antigen L, a new antigen from tubercle bacilli active in delayed hypersensitivity in animals sensitized with living bacilli (B.C.G.)]. / L'antigène L, nouvel antigène tuberculeux actif en réaction d'hypersensibilité retardée chez l'animal sensibilisé par des bacilles vivants (Bacille B.C.G.).

This report described the occurrence of an antigen in the tubercle bacilli. The antigen, designated antigen L, was mainly active in delayed hypersensitivity reaction in Guinea Pigs sensitized with living bacilli such as the B.C.G. strain. It was found in cultures filtrates of bovine strains as well as the human strains of tubercle bacilli.

[Different pathogenic power of the original BCG strain of the Pasteur Institute and of an isoniazid resistant strain. Histopathological study and significance of local abscesses after inoculation of guinea pig]. / Pouvoir pathogène différent de la souche de BCG historique de l'Institut Pasteur et d'une souche isoniazido-résistante. Etude histopathologique et signification des abcès locaux après inoculation au cobaye

The inoculation into guinea pigs of 10 mg of BCG, obtained either from the Pasteur Institute original strain, or from a mutant isoniazid resistant strain, B1 catalase positive, caused in 40.4% of cases, the onset of abscesses at the point of injection. The frequency and course of these abscesses appear quite different with the two strains, 92.8% with the classical strain, persisting in more than half of the cases after one year, and 23.5% with the INH-resistant strains which regressed within 6 months. Histologically, the structure of the recent abscesses, with a large histiocytic wall often rich in bacteria, and a very cellular necrotic content, appears different from that of old abscesses with a thin wall and a heterogeneous content, calcification with reaction to foreign bodies, and presence of granules due to the persistance of bacteria. The INH-resistant strain is thus in the guinea pig, less aggressive and more rapidly eliminated than the classical Pasteur strain. The course and morphology of abscesses in guinea pigs are very similar to certain old lesions of human tuberculosis and involve, in all strains, phenomena of cell immunity both general and local.

[Comparative dose-effect relationship of a tuberculin standard on guinea pig sensitivity, using living and dead tuberculosis bacilli]. / Loe dose-effet comparative d'une tuberculine étalon sur cobayes sensibilisés soit par bacilles tuberculeux vivants, soit par bacilles tués

The sensitization of guinea pigs utilized for tuberculin titration may be obtained by a BCG primo-vaccination followed by an inoculation of live, virulent Myc. tuberculosis. This method, which results in a sub-evolutive form of tuberculosis, gives a satisfactory sensitization level but has the disadvantage of utilizing animals which are germ carriers and therefore dangerous. A method has been tested employing killed germs (Jamaica strain) coated with Freund's complete adjuvant H37Ra which utilizes animals presenting no danger in handling. The results of 105 titrations made on 453 guinea pigs during two years show that the reactions obtained are clear, easy to read and remain stable over a long period (F equals 0.1). The comparison of values thus obtained shows that in the case of killed bacilli in relation to live bacilli: the mean dimensions of the reaction with 5, 10 and 20 IU are significantly more important (P is less than 0.0001) and the dose-response slope is significantly steeper (P is less than 0.01) which offers more precise results. Sensitization by killed germs therefore offers the double advantage of handling non-dangerous animals and obtaining results which are more precise and easier to read.

Wax D from different bovine strains of Mycobacterium.

Wax D(P), a peptido-glycolipid found in extracts of Mycobacterium tuberculosis var. hominis, was not found in extracts of three strains of still-grown M. tuberculosis var. bovis (BCG, Marmorek and Dupray). However, extracts from three other bovine strains (Behring, LA and BB) did yield waxes D(P), and these did not differ as to their molar ratios of alanine/glutamic acid/diaminopimelic acid from human waxes D(P).