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We report the cloning and sequencing of the gene coding for antigen 88 from Mycobacterium tuberculosis by using monoclonal antibodies to screen an expression library in lambda gt11. The gene encodes a 403-amino-acid-residue protein with a calculated molecular mass of 43,790 Da which contains seven putative transmembrane alpha-helical domains and presents a significant homology to the PstA protein of Escherichia coli. In its N-terminal region, it contains a 61-amino-acid region highly homologous to the fifth transmembrane helix of E. coli PstC. PstA and PstC are the two hydrophobic subunits of an E. coli periplasmic phosphate permease. Since the phosphate-binding subunit of this putative permease in M. tuberculosis has previously been characterized, i.e., the 38-kDa mycobacterial protein (also called protein antigen b, Ag 5, and Ag 78) homologous to PstS of E. coli, it seems likely that functional permeases analogous to the periplasmic permeases of gram-negative bacteria also exist in mycobacteria.

DNA plasmids encoding Mycobacterium tuberculosis antigen 85 (Ag85) were tested as vaccines in animal models. Ag85 DNA induced relevant immune responses (i.e. T helper (Th) cells, Th1 cytokines and cytotoxic T lymphocytes) and was protective in mouse and guinea pig models of mycobacterial disease. Therefore, DNA vaccination holds promise as an effective means of preventing tuberculosis in humans. Furthermore, this technique is amenable to identifying the protective antigens of M. tuberculosis.

Using culture filtrate Ag-specific mAbs generated from mycobacteria-infected H-2b haplotype mice, we have previously identified three genes in the Mycobacterium tuberculosis genome, encoding proteins homologous to the periplasmic ATP-binding cassette phosphate-binding receptor PstS of the phosphate-specific transport system of E. coli. To define the potential vaccinal properties of these phosphate-binding proteins, female C57BL/6 mice were injected i.m. with plasmid DNA encoding PstS-1, PstS-2, or PstS-3 proteins from M. tuberculosis and immunogenicity and protective efficacy against i.v. challenge with M. tuberculosis H37Rv was analyzed. Significant levels of highly Ag-specific Abs and Th1-type cytokines IL-2 and IFN-γ could be detected following vaccination with each of the three genes. However, only mice vaccinated with PstS-3 DNA demonstrated significant and sustained reduction in bacterial CFU numbers in spleen and lungs for 3 mo after M. tuberculosis challenge, as compared with CFU counts in mice vaccinated with control DNA. Vaccination with PstS-2 DNA induced a modest reduction in CFU counts in spleen only, whereas vaccination with PstS-1 DNA was completely ineffective in reducing bacterial multiplication. In conclusion, our results indicate that DNA vaccination is a powerful and easy method for comparative screening of potentially protective Ags from M. tuberculosis and that the PstS-3 protein is a promising new subunit vaccine candidate.

DNA vaccines have been demonstrated to be effective in inducing protective cell-mediated immune responses in animal models of infectious disease. In order to investigate this approach for potential use as a vaccine for tuberculosis, DNA constructs encoding Mycobacterium tuberculosis antigen 85A (Ag85A) were prepared. Expression of Ag85A in mammalian cells was demonstrated by transient transfection of cells in vitro. Intramuscular injection of Ag85A DNA vaccines resulted in the generation of anti-Ag85A antibodies and robust cell-mediated immune responses, as measured by lymphoproliferation of spleen cells in vitro upon specific antigen restimulation, leading to protection in animal challenge models. Therefore, the technique of DNA vaccination is effective in inducing relevant immune responses for protection against tuberculosis and may be used to identify the protective antigens of M. tuberculosis.

DNA vaccination is an effective means of inducing both humoral and cell-mediated immunity in animal models of infectious disease. Presented here are data generated in two distinct disease models; one viral (influenza) and one bacterial (tuberculosis). Specifically, plasmid DNA encoding an influenza virus antigen (nucleoprotein; NP) and a Mycobacterium tuberculosis antigen (antigen 85; Ag85) were prepared and tested as DNA vaccines in mice. In both cases, high titer antibody responses and robust cell-mediated immune responses were induced against the respective antigens. With respect to the latter, lymphocyte proliferation, Th1-type cytokine secretion, and cytotoxic T lymphocyte responses were observed upon restimulation with antigen in vitro. Furthermore, protective efficacy in animal challenge models was demonstrated in both systems. The data support the hypothesis that DNA vaccination will prove to be a broadly applicable technique for inducing immunity against various infectious diseases.

The phenomenon of acute phase (AP) response can be reproduced in vitro using cultured cells of hepatic origin by stimulation with the crude supernatant of activated monocytes (MoCM). Several monocyte-derived factors have been identified which might be responsible, alone or in combination, for the induction of AP response, but recently the attention has been focused on interleukin 6 (IL-6). We have previously shown that part of the AP response consists of the increase in the rate of transcription of the AP genes. Here we have treated the human hepatoma cell line Hep 3B with either crude MoCM or recombinant IL-6 and compared the effect of the two stimulants on the expression of both endogenous AP genes and recombinant plasmids introduced into the cells by transfection. The transfected plasmids contained the 5'-flanking region of AP genes fused to the coding region of the bacterial chloramphenicol acetyltransferase gene. We observe that the induction of mRNA accumulation of the endogenous genes corresponds to the transcriptional activation of the chloramphenicol acetyltransferase fusions. This is good evidence that the effect of IL-6 is totally or partially exerted at the level of transcription and that short segments of the 5'-flanking sequences of the inducible genes contain IL-6-responsive elements. Our results show that IL-6 is fully effective only on some of all the genes induced or repressed by MoCM, whereas others are only partially affected or totally nonresponsive.

Neutralizing monoclonal antibodies specific for human interleukin-6 (IL-6) bind two distinct sites on the IL-6 protein (sites I and II). Their interference with IL-6 receptor binding suggested that site I is a receptor-binding site of IL-6, whereas site II is important for signal transduction. Mutagenesis of site II could therefore result in the isolation of IL-6 receptor antagonists. To test this hypothesis, a panel of IL-6 mutant proteins was constructed that did not bind to a site II-specific monoclonal antibody. One such site II mutant protein (with double substitution of Gln-160 with Glu and Thr-163 with Pro) was found to be an antagonist of human IL-6. It was inactive on human CESS cells, weakly active on human HepG2 cells, but active on mouse B9 cells. It could specifically antagonize the activity of wild-type IL-6 on CESS and HepG2 cells. The binding affinity of this variant for the 80-kDa IL-6 receptor was similar to that of wild-type IL-6. High affinity binding to CESS cells, however, was abolished, suggesting that the mutant protein is inactive because the complex of the 80-kDa IL-6 receptor and the mutant protein cannot associate with the signal transducer gp130. The human IL-6 antagonist protein may be potentially useful as a therapeutic agent.

A mycobacterial expression-secretion vector was constructed in which the Escherichia coli alkaline phosphatase (phoA) reporter gene was placed under the control of the Mycobacterium tuberculosis 85A promoter and secretion signal sequences. In recombinant Mycobacterium smegmatis and Mycobacterium bovis BCG, PhoA activity could readily be detected on the mycobacterial cell surface and in the culture supernatant, indicating that the 85A signals can drive heterologous expression and secretion in both species. In contrast to the mycobacteria, the 85A promoter did not function in E. coli. We mapped the promoter region by progressive deletions using BAL 31 exonuclease and by primer extension analysis. Insertion and deletion mutations within the promoter region indicated that, unlike most E. coli promoters but similar to Streptomyces promoters, the position of the putative -35 region was not critical for efficient promoter activity. In addition, we investigated the ability of the identified signals to drive the production and secretion in BCG of recombinant Schistosoma mansoni glutathione S-transferase (Sm28GST), a protective antigen against schistosomiasis. BALB/c mice immunized with the recombinant BCG by a single dose exhibited a weak but specific T-cell response to Sm28GST.