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whole bacteria (e.g., H. pylori), whole viruses (e.g., influenza, Sendai, measles), and even whole protozoa (e.g., Toxoplasma gondii). Unlike LPS, CT and the antigen have to be administered together by the mucosal route, and in mice this usually requires microgranules. But all the properties of CT as an immunogen seem to rub off on the accompanying antigen, notably a prolonged memory response. Researchers have also succeeded in attaching peptide antigens directly to the CT molecule, with similar results.

Adjuvant Action of Cholera Toxin. CT has stimulatory effects on macrophages for IL-6 production, about a 30-fold increase. LPS has a somewhat greater effect, but the two together have even greater effect than either alone. On the other hand, CT does not stimulate TNF production, and it inhibits the stimulatory effect of LPS on TNF production.

Researchers have also achieved interesting effects by varying the sequence in which antigen-presenting cells are stimulated. Using bone marrow macrophages grown in vitro with M-CSF, for example, they found that both IFN and CT stimulate IL-6, and the combination stimulates better than either alone. But when the macrophages are “primed” with IFN for as little as 30 minutes before CT is introduced, there is an enormous increase in IL-6 production. This finding suggests that the sequence in which the immune system is stimulated may be more important than the mixture of stimulants.

Researchers are finding similar effects on production of the costimulatory molecule B7: CT alone stimulates 1.7 percent to 10.3 percent of cells to express B7, while CT and IFN together stimulate 40 percent (as measured by flow cytometry). Further analysis shows that B7 comes in two varieties, B7–1 (thought to preferentially stimulate the Th1 pathway) and B7–2 (thought to stimulate the Th2 pathway). B7–1 remains fairly constant regardless of stimulus, while B7–2 increases from 4 percent to 20 percent with CT, to 20 percent with IFN, to 56 percent with CT and IFN together. In short, CT appears to have a preferential, up-regulation effect on B7–2, the molecule that appears to be responsible for the costimulatory effect of CT and IFN. CT produces these effects down to the nanogram range, so it is a potent, very-low-dose effect of CT.

In vivo, the net effect of cholera toxin is to enhance T-cell priming, probably through its effects on macrophages. A single injection of CT plus KLH produces enhanced proliferation in response to KLH from multiple tissues, with cells producing a variety of cytokines for both the Th1 and Th2 pathways. CT also increases precursor cell frequency from 1:23,000 to 1:900. The net effect of all these biologic effects is strong stimulation.

There is a single dominant epitope for T cells on the CT molecule, a peptide designated CTV–89–100. Experiments with this peptide in mice have shown that when the animal is tolerized to CT as an antigen, CT loses its adjuvant effect—both the T-cell and B-cell responses were blocked. Other experiments have shown that the adjuvant effect is also blocked unless both the A subunit and B subunit of the CT molecule are present.

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