The Journal of Experimental Medicine
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© The Rockefeller University Press, 0022-1007/1997/4/1203/ $5.00
The Journal of Experimental Medicine, Volume 185, Number 7, April 7, 1997 1203-1210

Article

Mutants in the ADP-ribosyltransferase Cleft of Cholera Toxin Lack Diarrheagenicity but Retain Adjuvanticity

Shingo Yamamoto*,{ddagger}, Yoshifumi Takeda{ddagger},§, Masafumi Yamamoto*, Hisao Kurazono*, Koichi Imaoka*, Miho Yamamoto*, Kohtaro Fujihashi*, Masatoshi Noda||, Hiroshi Kiyono*,¶, and Jerry R. McGhee*

From the * Immunobiology Vaccine Center and Department of Microbiology and Department of Oral Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294; {ddagger} Department of Microbiology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan; § Research Institute, International Medical Center of Japan, Shinjuku-ku, Tokyo 162, Japan; || Second Department of Microbiology, Faculty of Medicine, Chiba University, Chuo-ku, Chiba 260, Japan; and Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565, Japan

Cholera toxin (CT), the most commonly used mucosal adjuvant in experimental animals, is unsuitable for humans because of potent diarrhea-inducing properties. We have constructed two CT-A subunit mutants, e.g., serine-> phenylalanine at position 61 (S61F), and glutamic acid-> lysine at 112 (E112K) by site-directed mutagenesis. Neither mutant CT (mCT), in contrast to native CT (nCT), induced adenosine diphosphate-ribosylation, cyclic adenosine monophosphate formation, or fluid accumulation in ligated mouse ileal loops. Both mCTs retained adjuvant properties, since mice given ovalbumin (OVA) subcutaneously with mCTs or nCT, but not OVA alone developed high-titered serum anti-OVA immunoglobulin G (IgG) antibodies (Abs) which were largely of IgG1 and IgG2b subclasses. Although nCT induced brisk IgE Ab responses, both mCTs elicited lower anti-OVA IgE Abs. OVA-specific CD4+ T cells were induced by nCT and by mCTs, and quantitative analysis of secreted cytokines and mRNA revealed a T helper cell 2 (Th2)-type response. These results now show that the toxic properties of CT can be separated from adjuvanticity, and the mCTs induce Ab responses via a Th2 cell pathway.

Address correspondence to Dr. Jerry R. McGhee, Immunobiology Vaccine Center and the Department of Microbiology, University of Alabama at Birmingham, 845 19th Street South, Birmingham, Alabama 352942170.

1Abbreviations used in this paper: AFC, antibody-forming cell; CHO, Chinese hamster ovary; CT, cholera toxin; ELISPOT, enzyme-linked immunospot; LT, heat-labile toxin; m, mutant; n, native; r, recombinant; RT, reverse transcriptase; S, secretory.


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