Mycobacterial heat-shock proteins as carrier molecules.

We have previously shown that the priming of mice with live Mycobacterium tuberculosis var. bovis (Bacillus Calmette-Guérin, BCG) and immunization with the repetitive malaria synthetic peptide (NANP)40 conjugated to purified protein derivative (PPD), led to the induction of high and long-lasting titers of anti-peptide IgG antibodies, overcoming the requirement of adjuvants and the genetic restriction of the antibody response to the peptide (Lussow et al., Proc. Natl. Acad. Sci. USA 1990. 87:2960). This initial work led us to the following observations. BCG had to be live for priming to lead to the induction of anti-peptide antibodies. Surprisingly, priming with other living microorganisms which chronically infect the macrophage (e.g. Salmonella typhimurium and Leishmania major) also induced anti-peptide antibodies in mice immunized with PPD-(NANP)40 conjugate. It was, thus, hypothesized that molecules expressed during active infection and also known to be highly conserved between species, namely the heat-shock proteins (hsp), could mediate the T cell sensitization required for the production of anti-peptide antibodies. In fact, when the PPD protion of the conjugate was replaced by a highly purified recombinant protein corresponding to the 65-kDa (GroEL-type) hsp of M. bovis, this resulted in the production of anti-(NANP) IgG antibodies in BCG-primed mice, irrespective of the major histocompatibility complex-controlled responsiveness to the (NANP) sequence itself. Further, similar induction of anti-peptide antibody response was also obtained with a recombinant 70-kDa (DnaK-type) hsp of M. tuberculosis, but not with a small molecular mass (18 kDa) of M. leprae. Finally, an adjuvant-free carrier effect for anti-peptide IgG antibody production in BCG-primed mice, was also exerted by the GroEL hsp of Escherichia coli. This finding that hsp can act as carrier molecules without requiring conventional adjuvants is of potential importance in the development of vaccine strategies.

Identification of components of IC purified from human sera. II. Demonstration of mycobacterial antigens in immune complexes isolated from sera of lepromatous patients.

Immune complexes have been purified from sera of patients with lepromatous leprosy, using solid phase conglutinin and analysed by SDS-PAGE. Some of their components have been immunologically identified after electrophoretic blotting on nitrocellulose. First, immunoglobulins, complement components (C1q, C1s, C3) and CRP were found in IC. Secondly, one mycobacterial antigen of 67 kD was directly identified in IC while two other components (20 kD and 14.4 kD) of possible M. leprae origin were also found in IC. This study suggests that lepromatous patients develop a good antibody response against some M. leprae antigens (33 kD and 12 kD), which are rapidly eliminated from circulation, while other M. leprae antigens (e.g. 67 kD) can persist in relative antigen excess, within circulating IC.

SDS-PAGE analysis of M. leprae protein antigens reacting with antibodies from sera from lepromatous patients and infected armadillos.

Studies have been conducted to characterize M. leprae bacilli derived from infected armadillos. First, the proteins of the mycobacterial extracts were fractionated by SDS-PAGE. Subsequently, the proteins in the gel were electrophoretically transferred on a strip of nitrocellulose paper by the technique of 'electrophoretic blotting'. The separated bacterial protein bands, thus immobilized on the nitrocellulose paper were made to react immunologically with sera from the lepromatous patients, infected armadillo sera and other experimental mycobacterial antisera. It was observed that a majority of M. leprae proteins contained antigenic determinants also present on proteins of BCG. In addition, only two specific antigen bands of 33KD and 12KD were conspicuously detected by the patients' sera and the infected armadillo sera. These substances were further identified as polysaccharides or glycoproteins since they could only be stained by Schiff's reagent or alcian blue. Only 12KD glycoprotein band reacted with concanavalin A, whereas wheat germ agglutinin (WGA) did not show any reaction with them. These 33KD and 12KD glycoprotein antigens were found to lose their antigenicity after pepsin treatment and can be considered as glycoproteins. Further, radiolabelling experiments showed that 12KD antigen underwent radioiodination under usual conditions, but 33KD glycoprotein failed to be similarly radiolabelled. It is suggested that these protein antigens have M. leprae specific determinants on a cross-reacting component.

Immune complexes and complement hypercatabolism in patients with leprosy.

The occurrence of immune complexes in the serum and the level of the C3 breakdown product C3d in the plasma from patients with leprosy were studied by quantitative methods and the results were compared in various forms of the disease. These studies were performed on sixty-two samples from twenty-six patients. The serum 125I-C1q binding activity was found to be increased by more than 2 s.d., as compared to the normal values, in most of the sera from patients with erythema nodosum leprosum (ENL) (80%) and uncomplicated lepromatous leprosy (82%), but also in the sera from patients with tuberculoid leprosy (58%). In vitro studies suggested that immune complexes involving mycobacterial antigens were present in leprosy sera. An increased C3d level (greater than 2s.d.) was also found in most of the plasma from patients with ENL (70%), but rarely in the plasma from patients with uncomplicated lepromatous leprosy (18%) and never in tuberculoid leprosy patients' plasma. The absence of a significant correlation between the 125I-C1q binding activity and the C3d level in leprosy patients may suggest that extravascular immune complexes are involved in the complement activation occurring in ENL. The quantitation of C3d in plasma may be of some practical interest in the early diagnosis of ENL complications of leprosy.