Twenty-one patients treated for active tuberculosis were examined for immune reactivity to purified protein derivative (PPD) and to a purified 32-kDa protein antigen (P32) from Mycobacterium bovis, strain BCG. Lymphoproliferation of peripheral blood leucocytes to PPD and P32 was positive in 95% and 71% of the patients respectively. A positive IFN-gamma response was detected in 62% against PPD and in 48% against P32. Low blastogenesis and IFN-gamma production were observed, especially in patients with poor general health and advanced tuberculous lesions. Twelve out of twelve (100%) of the tuberculin-positive healthy volunteers responded to PPD and P32 with mean lymphoproliferation and IFN-gamma values that were higher than in the patient group. Twelve tuberculin-negative control subjects were completely unreactive to PPD and P32 antigen. On the other hand, IgG antibodies in the serum were detected in 95% of the patients against PPD, in 77% of the patients against P32 but in none of the tuberculin-positive or negative healthy volunteers. The highest IgG levels against PPD were found in those patients with the lowest in vitro lymphoproliferation and IFN-gamma production (r = -0.54; P less than 0.05). Nonspecific interferon production following induction with Newcastle disease virus, Corynebacterium parvum, or phytohaemagglutinin was comparable in the control and patient groups. Finally, low IFN-alpha titres were detected in the serum of about 50% of the patients.

An immunogenic and skin-reactive protein called P64 was purified from Sauton zinc-deficient culture filtrate of Mycobacterium bovis BCG by using successively hydrophobic chromatography on phenyl-Sepharose, ion exchange on DEAE-Sephacel, and molecular sieving on Sephadex G-200. The final P64 preparation was found to be homogeneous based on several analyses. Protein P64 was a constituent of BCG cells since it was present in soluble cellular extract from normally grown BCG cells. It represented 8 to 9% of the soluble proteins of the extract and appeared as the major soluble protein antigen of BCG. This protein was found to have a molecular weight of 64,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but in molecular sieving it eluted at a volume corresponding to a molecular weight of 246,000. An abnormal UV spectrum was observed for this protein. Its amino acid composition showed an abundance of acidic amino acids (or their amides). Aromatic amino acids represented only 3% of the total amino acid residues. The NH2-terminal amino acid sequence of this protein (10 amino acids) was determined. Its sugar content measured with the phenol-sulfuric acid test was lower than 0.3% (wt/wt). Isolated P64 was tested by various crossed-immunoelectrophoresis techniques and was shown to correspond to antigen 82 in the reference system for BCG antigens. The protein antigen P64 elicited a delayed cutaneous reaction in guinea pigs sensitized with either living or heat-killed BCG. Its potency in skin reaction was, respectively, two-and threefold that of the BCG purified protein derivative. The two types of sensitization used for skin test reactions promoted significant immunoglobulin G antibody production against the protein antigen P64 in guinea pigs 7 weeks after sensitization.

After growth of six strains of mycobacteria on Sauton medium in the absence of added Zn2+, cell yields were lowered, to between 22% and 67% of the yields obtained when Zn2+ (5 microM) was added. Two immunodominant proteins, named P64 and P32 (antigens of 62-65 kDa and 29-33 kDa, respectively) were abundant in culture filtrates after growth of mycobacteria. P64 was present at elevated concentrations (showing a 9- to 16-fold increase as a percentage of the total protein released) after Zn2+-deficient growth of five of the six strains studied; in Mycobacterium tuberculosis it represented 25% of all released proteins. However, little P64 was detected in culture filtrates of M. fortuitum and of M. phlei grown under Zn2+ deficiency, and in the latter there was no increase of P64 during Zn2+ deficiency.