Downloaded from www.microbiologyresearch.org by IP: 54.242.161.225 On: Sun, 17 Apr 2016 20:33:54 Journal of General Microbiology (1993), 139, 1543-1549. Printed in Great Britain 1543 Immunological and functional characterization of proteins of the Mycobacterium tuberculosis antigen 85 complex using synthetic peptides S. C. LE~o,~* J. D. LOPES~ and M. E. PATARROYO' ' Instituto de Inmunologia - Hospital Sun Juan de Dios, Universidad Nacional de Colombia, Avenida la 10-01, Bogoth, Colombia 2Disciplina de Biologia Celular, Escola Paulista de Medicina, Rua Botucatu, 862 80, 04023-062 SZo Paulo, Brazil (Received 10 November 1992; revised 16 February 1993; accepted 3 March 1993) As tuberculosis reemerges as an important health problem worldwide, new drugs, better diagnostic tests and vaccines are being sought. In order to identify potentially useful peptides for the development of a synthetic vaccine against tuberculosis, immunological and functional studies were performed using proteins of the antigen 85 complex. Western blot (immuno-blot) analysis and a lymphoproliferation study was used to investigate the B- and T-cell immune response of tuberculosis patients, healthy household contacts and normal controls to proteins of the Mycobacterium tuberculosis antigen 85 complex. Peptides derived from the 85A amino acid sequence were synthesized and used in fibronectin-binding and in ELISA assays. A peptide with the sequence CQPACRKAGCQTYKWEC bound to radiolabelled fibronectin in a time-dependent manner and was recognized by human sera in ELISA. This peptide was identified as a potential component of a synthetic vaccine against tuberculosis . Introduction The antigen 85 complex is a group of related myco- bacterial proteins actively secreted into culture fluids. Three major components have been described by different authors and assigned different names. The P32 antigen described by De Bruyn et al. (1987) and by Borremanns et al. (1989), as well as MPT44 and MPB44 from Nagai's classification of major mycobacterial culture fluid proteins (Nagai et al., 1981) correspond to antigen 85A of the classification proposed by Wiker et al. (1986~). Both the a antigen cloned and sequenced by Matsuo et al. (1988) and the MPB59 protein, character- ized by Wiker et al. (1986b), correspond to 85B. Finally, the MPT45 and MPB45 antigens correspond to 85C (Nagai et al., 1991). In spite of the published data regarding the immuno- logical response of tuberculosis, leprosy and AIDS patients to antigens of the 85 complex (Huygen et al., 1988; Rumschlag et al., 1988; Drabick & Baker, 1990), the role these antigens play in the development of clinical * Author for correspondence.Tel. 57 1 233 9006; fax 57 1 280 3999. Abbreviations: BCI/NBT, 5-bromo-4-chloro-3-indolyl phosphate/ nitroblue tetrazolium; FN, fibronectin. disease or protective immunity in humans is not yet clearly understood. Components of antigen 85 complex were shown to be located in the outer cell wall and on the cell surface of Mycobacterium tuberculosis bacilli (Abou-Zeid et al., 1991 ; Rambukkana et al., 1991) and to participate in the binding of mycobacteria to fibronectin (FN)-coated surfaces (Ratliff et al., 1988). The binding capacity of antigen 85 to F N could have clinical as well as protective significance. The phagocytosis of mycobacteria by monocytes/ macrophages could be influenced by the interaction between antigen 85 on the surface of mycobacteria, plasma FN, and FN receptors on the surface of the monocyte/macrophage. An immunological response against the 85 complex could interfere in this interaction, affecting the phagocytosis of the bacilli. Although antibodies are not considered to participate in the protective immunity against tuberculosis, it is possible that some degree of protection could be afforded by antibodies that effectively block the interaction between antigen 85 and FN. The fact that immunization with live mycobacterial vaccine efficiently generates a protective response, while killed preparations do not (Orme, 1988), is further evidence that proteins released by live bacilli are 0001-7942 0 1993 SGM