The effects of heterologous gene dosage as well as Salmonella typhimurium strain variability on immune response toward both the heterologous antigen, the nontoxic mutant of the Escherichia coli heat- labile enterotoxin LTK63, and the carrier Salmonella strain have been analyzed. Effects of a single integration into the host DNA and different- copy-number episomal vectors were compared in S. typhimurium Δcya Δcrp Δasd strains of two different serotypes, UK-1 and SR-11. Expression of the enterotoxin in the different Salmonella isolates in vitro was found to vary considerably and, for the episomal vectors, to correlate with the plasmid copy number. LTK63-specific serum immunoglobulin G (IgG) and mucosal immunoglobulin A (IgA) antibodies were highest in mice immunized with the high-level-expression strain. High anti-LTK63 IgG and IgA titers were found to correspond to higher anti-Salmonella immunity, suggesting that LTK63 exerts an adjuvant effect on response to the carrier. Statistically significant differences in anti-LTK63 immune response were observed between groups of mice immunized with the attenuated Δcya Δcrp UK-1 and SR-11 derivatives producing the antigen at the same rate. These data indicate that the same attenuation in S. typhimurium strains of different genetic backgrounds can influence significantly the immune response toward the heterologous antigen. Moreover, delivery of the LTK63 enterotoxin to the immune system by attenuated S. typhimurium strains is effective only when synthesis of the antigen is very high during the initial phase of invasion, while persistence of the S. typhimurium strain in deep tissues has only marginal influence.661224231Bost, K.L., Clements, J.D., In vivo induction of interleukin-12 mRNA expression after oral immunization with Salmonella dublin or the B subunit of Escherichia coli heat-labile enterotoxin (1995) Infect. Immun., 63, pp. 1076-1083Brocchi, M., Covone, M.G., Palla, E., Galeotti, C.L., Unpublished dataCárdenas, L., Clements, J.D., Stability, immunogenicity and expression of foreign antigens in bacterial vaccine vectors (1993) Vaccine, 11, pp. 126-135Cárdenas, L., Dasgupta, U., Clements, J.D., Influence of strain viability and antigen dose on the use of attenuated mutants of Salmonella as vaccine carriers (1994) Vaccine, 12, pp. 833-840Chatfield, S.N., Fairweather, N., Charles, I., Pickard, D., Levine, M., Hone, D., Posada, M., Dougan, G., Construction of a genetically defined Salmonella typhi Ty2 aroA, aroC mutant for the engineering of a candidate oral typhoid-tetanus vaccine (1992) Vaccine, 10, pp. 53-60Curtiss III, R., Kelly, S.M., Salmonella typhimurium deletion mutants lacking adenylate cyclase and cyclic AMP receptor protein are avirulent and immunogenic (1987) Infect. Immun., 55, pp. 3035-3043Curtiss III, R., Kelly, S.M., Gulig, P.A., Nakayama, K., Selective delivery of antigens by recombinant bacteria (1989) Curr. Top. Microbiol. Immunol., 146, pp. 35-119Curtiss III, R., Attenuated Salmonella strains as live vectors for the expression of foreign antigens (1990) New Generation Vaccines, pp. 161-188. , G. C. Woodrow and M. M. Levine (ed.), Marcel Dekker, Inc., New York, N.YDallas, W.S., Falkow, S., Amino acid sequence homology between cholera toxin and Escherichia coli heat-labile toxin (1980) Nature, 288, pp. 499-501Di Tommaso, A., Saletti, G., Pizza, M., Rappuoli, R., Dougan, G., Abrignani, S., Douce, G., De Magistris, M.T., Induction of antigen-specific antibodies in vaginal secretions by using a nontoxic mutant of heat-labile enterotoxin as a mucosal adjuvant (1996) Infect. Immun., 64, pp. 974-979Dougan, G., The molecular basis for the virulence of bacterial pathogens: Implications for oral vaccine development (1994) Microbiology, 140, pp. 215-224Dougan, G., Chatfield, S.N., Pickard, D., O'Caliaghan, D., Maskell, D., Construction and characterization of Salmonella vaccine strains harbouring mutations in two different aro genes (1988) J. Infect. Dis., 158, pp. 1329-1335Galan, J.E., Nakayama, K., Curtiss III, R., Cloning and characterization of the asd gene of Salmonella typhimurium: Use in stable maintenance of recombinant plasmids in Salmonella typhimurium (1990) Gene, 94, pp. 29-35Gentry-Weeks, C.R., Hultsch, A.-L., Kelly, S.M., Keith, J.M., Curtiss III, R., Cloning and sequencing of a gene encoding a 21-kilodalton outer membrane protein from Bordetella avium and expression of the gene in Salmonella typhimurium (1992) J. Bacteriol., 174, pp. 7729-7742Gyles, C.L., Barnum, D.A., A heat-labile enterotoxin from strains of E. coli enteropathogenic for pigs (1969) J. Infect. Dis., 120, pp. 419-426Hofstra, H., Witholt, B.J., Kinetics of synthesis, processing and membrane transport of heat-labile enterotoxin, a periplasmic protein in E. coli (1984) J. Biol. Chem., 259, pp. 15182-15187Hoiseth, S.K., Stocker, B.A.D., Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines (1981) Nature, 291, pp. 238-239Hone, D., Morona, R., Attridge, S., Hackett, J., Construction of a defined galE mutant of Salmonella for vaccine use (1987) J. Infect. Dis., 156, pp. 167-174Hone, D., Harris, A.M., Chatfield, S., Dougan, G., Levine, M.M., Construction of genetically-defined double aro mutants of Salmonella typhi (1991) Vaccine, 9, pp. 810-816Karem, K.L., Chatfield, S., Kuklin, N., Rouse, B.T., Differential induction of carrier antigen-specific immunity by Salmonella typhimurium live-vaccine strains after single mucosal or intravenous immunization of BALB/c mice (1995) Infect. Immun., 63, pp. 4557-4563Magagnoli, C., Manetti, R., Fontana, M.R., Giannelli, V., Giuliani, M.M., Rappuoli, R., Pizza, M., Mutations in the A subunit affect yield, stability, and protease sensitivity of nontoxic derivatives of heat-labile enterotoxin (1996) Infect. Immun., 64, pp. 5434-5438Masked, D.J., Sweeney, K.J., O'Callaghan, D., Hormaeche, C.E., Liew, F.Y., Dougan, G., Salmonella typhimurium aroA mutants as carriers of the Escherichia coli heat-labile enterotoxin B subunit to the murine secretory and systemic immune system (1987) Microb. Pathog., 2, pp. 211-221Miller, S.I., Loomis, W.P., Alpuche-Aranda, C., Behlau, I., Hohmann, E., The PhoP virulence regulon and live oral Salmonella vaccines (1993) Vaccine, 11, pp. 122-125Nakayama, K., Kelly, S.M., Curtiss III, R., Construction of an ASD+ expression-cloning vector: Stable maintenance and high level expression of cloned genes in a Salmonella vaccine strain (1988) Bio/Technology, 6, pp. 693-697Oyston, P.C.F., Williamson, E.D., Leary, S.E.C., Eley, S.M., Griffin, K.F., Titball, R.W., Immunization with live recombinant Salmonella typhimurium aroA producing F1 antigen protects against plague (1995) Infect. Immun., 63, pp. 563-568Packard, D., Li, J., Roberts, M., Maskell, D., Hone, D., Levine, M.M., Dougan, G., Chatfield, S., Characterization of defined ompR mutants of Salmonella typhi: OmpR is involved in the regulation of Vi polysaccharide expression (1994) Infect. Immun., 62, pp. 3984-3993Pizza, M., Domenighini, M., Hol, W., Giannelli, V., Fontana, M.R., Giuliani, M.M., Magagnoli, C., Rappuoli, R., Probing the structure-activity relationship of Escherichia coli LT-A by site-directed mutagenesis (1994) Mol. Microbiol., 14, pp. 51-60Pizza, M., Fontana, M.R., Giuliani, M.M., Domenighini, M., Magagnoli, C., Giannelli, V., Nucci, D., Rappuoli, R., A genetically detoxified derivative of heat-labile Escherichia coli enterotoxin induces neutralizing antibodies against the A subunit (1994) J. Exp. Med., 180, pp. 2147-2153Redman, T.K., Harmon, C.C., Lallone, R.L., Michalek, S.M., Oral immunization with recombinant Salmonella typhimurium expressing surface protein antigen A of Streptococcus sohrinus: Dose response and induction of protective humoral responses in rats (1995) Infect. Immun., 63, pp. 2004-2011Rossolini, G.M., Muscas, P., Chiesurin, A., Satta, G., Molecular cloning and expression in Escherichia coli of the Salmonella typhi gene cluster coding for type 1 fimbriac (1993) The Biology of Salmonella, pp. 408-412. , F. Cabello et al. (ed.), Plenum Publishing Co., New York, N.YSambrook, J., Fritsch, E.F., Maniatis, T., (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., , Cold Spring Harbor Laboratory, Cold Spring Harbor, N.YSanderson, K.E., Hessel, A., Rudd, K.E., Genetic map of Salmonella typhimurium, edition VIII (1995) Microbiol. Rev., 59, pp. 241-303Schödel, F., Kelly, S.M., Peterson, D.L., Milich, D.R., Curtiss III, R., Hybrid hepatitis B virus core-pre-S proteins synthesized in avirulent Salmonella typhimurium and Salmonella typhi for oral vaccination (1994) Infect. Immun., 62, pp. 1669-1676Smith, H.W., Halls, S., Escherichia coli enterotoxin (1967) J. Pathol. Bacteriol., 93, pp. 531-543Spicer, E.K., Kavanaugh, W.M., Dallas, W.S., Falkow, S., Konigsberg, W.H., Shafer, D., Sequence homologies between A subunits of E. coli and V. cholera enterotoxin (1981) Proc. Natl. Acad. Sci. USA, 78, pp. 50-54Staats, H.F., Jackson, R.J., Marinaro, M., Takahashi, I., Kiyono, H., McGhee, J.R., Mucosal immunity to infection with implications for vaccine development (1994) Curr. Opin. Immunol., 6, pp. 572-583Tacket, C.O., Hone, D.M., Curtiss III, R., Kelly, S.M., Losonsky, G., Guers, L., Harris, A.M., Levine, M.M., Comparison of the safety and immunogenicity of ΔaroC ΔaroD and Δcya Δcrp Salmonella typhi strains in adult volunteers (1992) Infect. Immun., 60, pp. 536-541Tsai, C.M., Frash, C.E., A sensitive stain for detecting lipopolysaccharides in polyacrylamide gels (1983) Anal. Biochem., 119, pp. 115-119Walker, R.I., New strategies for using mucosal vaccination to achieve more effective immunization (1994) Vaccine, 12, pp. 387-400Zhang, T., Stanley, S.L., Oral immunization with an attenuated vaccine strain of Salmonella typhimurium expressing the serine-rich Entamoeba histolytica protein induces an antiamebic immune response and protects gerbils from amebic liver abscess (1996) Infect. Immun., 64, pp. 1526-1531