Proteins that are actively secreted by Mycobacterium tuberculosis generate immune responses in the infected host. This has prompted the characterization of protein components of mycobacterial culture filtrates to develop subunit vaccines and immunodiagnostic reagents. Fractionation of filtrates of M. tuberculosis cultures has yielded an abundant protein called MPT63, which has an apparent molecular mass of 18 kDa. We report the molecular cloning and nucleotide sequence of the mpt63 gene, purification of recombinant MPT63 antigen from Escherichia coli cells, and serological characterization of MPT63. Nucleotide sequence analysis of mpt63 identified an open reading frame encoding a protein of 159 amino acids (aa) consisting of a 29-aa secretion signal peptide and a 130-aa mature MPT63 protein. Recombinant MPT63 protein, purified from E. coli cells, and native MPT63, purified from M. tuberculosis culture filtrates, were indistinguishable in serological assays. Thus, the recombinant protein constitutes a valuable reagent for immunological studies. MPT63 evoked humoral immune responses in guinea pigs infected with virulent M. tuberculosis by the aerosol route. The mpt63 gene is found only in species of the M. tuberculosis complex, as shown by DNA hybridization experiments. Moreover, polyclonal antibody against MPT63 does not cross-react with proteins of a common environmental mycobacterial species, Mycobacterium avium. The absence of cross-reactive epitopes makes MPT63 an attractive candidate as an M. tuberculosis complex-specific diagnostic reagent. In particular, evaluation of MPT63 as an M. tuberculosis complex-specific reagent for diagnostic skin testing is under way.

It has earlier been shown that the Plasmodium falciparum-reactive human monoclonal antibody 33G2 inhibits parasite growth in vitro as well as cytoadherence of infected red blood cells to melanoma cells in vitro. MoAb 33G2 recognizes an epitope of the P. falciparum antigen Ag332 and cross-reactive determinants in Pf155/RESA and Pf11.1 located in repetitive regions containing sequences of regularly spaced pairs of glutamic acid. To study whether antibodies of this specificity frequently occur in human immune sera and if they could be of importance for protective immunity, antibodies were affinity purified on MoAb 33G2 reactive Ag332 peptides. The epitope specificity of the affinity purified antibodies, determined by the Pepscan method, resembled that of MoAb 33G2, but showed differences in fine specificity. The antibodies cross-reacted to some extent with Pf11.1 and Pf155/RESA repeat peptides as detected by peptide ELISA and Pepscan. In indirect immunofluorescence all purified antibodies displayed a dotted pattern of staining of late stage infected red blood cells of two lines of the P. falciparum strain FCR3, including a Pf155/RESA deficient line. The in vitro growth of these two lines was efficiently inhibited by the affinity purified antibodies, indicating that their inhibitory effect was mainly due to reactivity with antigens other than Pf155/RESA. This, and the fact that Pf11.1 has been shown not to be expressed by the asexual stages suggests that Ag332 may be an important target for potentially protective antibodies in vivo and that Ag332 based immunogens are of interest for development of malaria subunit vaccines.

Immune responses to the repeat regions of the Plasmodium falciparum antigen Pf155/RESA have been extensively studied, and antibodies to the repeats are known to interfere with parasite growth both in vitro and in vivo. Less is known with regard to the effect on parasites of antibodies to the nonrepeat regions of the antigen. In the present study, rabbits were immunized with synthetic peptides corresponding to three different nonrepeated sequences of antigen Pf155/RESA. The reactivity of the antibodies with the particular peptides was analyzed by enzyme-linked immunosorbent assay (ELISA) and that with the parasite antigen, by immunoblotting and immunofluorescence. Although all antisera reacted strongly with the corresponding synthetic peptides, they reacted only weakly with full-length Pf155/RESA in either of the methods used. The specificity of the antibodies for Pf155/RESA was confirmed by their failure to stain Pf155/RESA-deficient parasites in erythrocyte membrane immunofluorescence, a method mainly detecting this antigen. Antibodies to the nonrepeat sequences also efficiently inhibited the merozoite invasion in vitro of Pf155/RESA+ parasites. However, these antibodies also inhibited Pf155/RESA-deficient parasites, indicating the presence of an antigen exhibiting a high degree of homology with Pf155/RESA. The results indicate that nonrepeat sequences of Pf155/RESA are immunogenic and may serve as targets for parasite-neutralizing antibodies, and, thus, the potential of the antigen as a vaccine candidate is emphasized.

SUMMARYAntigenic diversity in field populations of Plasmodium falciparum parasites may delay the acquisition of protective immunity to malaria, the development of which may thus require repeated exposure to infection over a prolonged period of time. In this study we show that P. falciparum parasites may vary in their sensitivity to antibody-mediated invasion/growth inhibition in vitro. Wild isolates of P. falciparum from children living in an endemic area of Burkina Faso were tested for their sensitivity to the growth inhibitory effects of antibodies originating from the same (autologous) and from other donors (heterologous). A significantly lower invasion inhibition activity was obtained when the isolates and antibodies were tested in autologous compared with heterologous combinations. The lower sensitivity to growth inhibition by autologous antibodies may be due to immune pressure in vivo, selecting from a heterogeneous parasite population those with a low expression of the antigens recognized by the host's antibodies. Alternatively, the parasites cultured from each child might represent expanding parasite populations, mainly constituting strains not earlier seen by the immune system of that specific host. The results reinforce the concern about Plasmodium antigenic diversity as a major obstacle towards the development of an effective malaria vaccine.

Antibodies to a non-repeat region of the Plasmodium falciparum antigen Pf155/RESA were investigated for their capacity to inhibit parasite cytoadherence to melanoma cells and parasite growth in vitro. The activities of these antibodies were studied since the target region in Pf155/RESA includes a cytoadherence-related motif also found in loop 3 and 7 of human erythrocyte band 3 protein. Overlapping multiple antigen peptides (MAPs) together spanning residues 199-220 of Pf155/RESA were used to raise antibodies in rabbits. Analysis of the fine specificity of these antibodies revealed that antibodies raised against largely overlapping sequences displayed highly different specificity patterns. Similarly, striking differences were seen when analysing the biological effect of antibodies to these MAPs. Antibodies to the cytoadherence-related motif of Pf155/RESA, as well as antibodies raised against a MAP based on a corresponding band 3 motif, inhibited cytoadherence but not parasite growth. In contrast, antibodies to sequences adjacent to the Pf155/RESA cytoadherence motif inhibited parasite growth in vitro but had no effect on cytoadherence.