Use of Mycobacterium tuberculosis complex-specific antigen cocktails for a skin test specific for tuberculosis

Abstract

The tuberculin skin test currently used to diagnose infection with Mycobacterium tuberculosis has poor diagnostic value, especially in geographic areas where the prevalence of tuberculosis is low or where the environmental burden of saprophytic, nontuberculous mycobacteria is high. Inaccuracy of the tuberculin skin test often reflects a low diagnostic specificity due to the presence in tuberculin of antigens shared by many mycobacterial species. Thus, a skin test specific for tuberculosis requires the development of new tuberculins consisting of antigens specific to M. tuberculosis. We have formulated cocktails of two to eight antigens of M. tuberculosis purified from recombinant Escherichia coli. Multiantigen cocktails were evaluated by skin testing guinea pigs sensitized with M. bovis BCG. Reactivity of multiantigen cocktails was greater than that of any single antigen. Cocktail activity increased with the number of antigens in the cocktail even when the same amount of total protein was used for cocktails and for each single antigen. A cocktail of four purified antigens specific for the M. tuberculosis complex elicited skin test responses only in BCG-immunized guinea pigs, not in control animals immunized with M. avium. These findings open the way to designing a multiantigen formulation for a skin test specific for tuberculosis.

FIG. 1

Skin test reactivity of purified recombinant antigens of M. tuberculosis tested singly and in combinations in guinea pigs immunized with M. bovis BCG. Six guinea pigs were sensitized as described in Materials and Methods. Five weeks after sensitization, animals were intradermally injected with 10 TU of PPD and 2 μg of purified recombinant antigens, singly or in combination. Results are expressed as the means (plus standard deviations) of the diameters of erythema measured 24 h after antigen injection. Ags 1–6, six antigens injected singly (1, MPT63; 2, MPT64; 3, MTC28; 4, MPT32; 5, MPT51; 6, 38 kDa); Combi1-2, two-antigen cocktail (antigens 1 and 2); Combi1-4, four-antigen cocktail (antigens 1 through 4); Combi1-6, six-antigen cocktail (antigens 1 through 6).

FIG. 2

Skin test reactivity of multiantigen cocktails in guinea pigs immunized with M. bovis BCG (solid symbols) and with M. avium (open symbols). Six guinea pigs were sensitized and skin tested as described in Materials and Methods. In this set of experiments, animals were skin tested with 10 TU of PPD and increasing amounts (0.5 to 8 μg) of multiantigen cocktails. Results are expressed as means of the diameters of erythema measured 24 h after antigen injection. Cocktail A, four M. tuberculosis complex-specific antigens (MPT63, MPT64, MTC28, and MPT70); cocktail B, four cross-reactive antigens (MPT51, Ag85B, MPT32, and KatG); cocktail C, eight-antigen cocktail (antigens in cocktails A plus B). ■, □, M. bovis PPD; ▴, ▵, M. avium PPD; •, ○, cocktails of purified antigens.

FIG. 3

Specificity for tuberculous mycobacteria of multiantigen cocktails tested at different doses. Protocols of guinea pig sensitization and skin testing with PPD and multiantigen cocktails were as described in the legend to Fig. 2. SpI was calculated as described in Table 2, footnote b. ⧫, PPD; ■, cocktail A (M. tuberculosis complex specific); ▴, cocktail B (cross-reactive); •, cocktail C (antigens in cocktails A plus B).