Splenocytes from naive mice and mice immunized with BRD509 alone did not produce detectable levels of IFN- upon in vitro stimulation with C fragment (Fig. traditional whole-cell killed vaccines (5, 9). The best-characterized of the live rationally attenuated salmonellae are those Phosphoramidon Disodium Salt harboring mutations in the prechorismate pathway (15). Prechorismate or mutant vaccines, as well as inducing protective immunity against virulent and and strain that harbors mutations in the genes and has been used extensively as a vaccine vector and for this reason has been included in our study (7, 16, 28, 31). The genes and genes. Similarly, the mutant has been investigated for its ability to carry heterologous antigens, although to a lesser extent. strains harbor a deletion in a serine Phosphoramidon Disodium Salt protease gene, and their avirulence may be due to their relative incapacity to mount a complete stress response. Chabalgoity et al. (3) successfully used the mutant as a vaccine vector and demonstrated protection against herpes simplex virus following immunization of mice with strains expressing a fusion protein comprising the C fragment of tetanus toxin and the glycoprotein D of herpes simplex virus. The antigen selected for our study has been extensively investigated in mutant expressing C fragment from the expression plasmid pTETtac4 successfully immunized mice against lethal challenge with TT. The aim of this study was to assess the capacity of a number of isogenic attenuated strains to act as vaccine vectors by correlating their ability to elicit immune responses with virulence, as measured by in vivo invasion and bacterial persistence. MATERIALS AND METHODS Bacterial strains and plasmids. The bacterial strains used in this study are described in Table ?Table1. Five1. Five of the six mutant strains studied are isogenic mutants of SL1344, whereas one, harboring the SR-11 background (Table ?(Table1.).1.). BRD175, BRD509, BRD578, BRD726, and LB5010 and pTETtac4 (10) were the generous gift of G. Dougan, Imperial College, London, England, and S. Chatfield and M. Roberts, Medeva Vaccine Research Unit, Imperial College. SL3261 was kindly supplied by B. A. D. Stocker (Stanford University, Palo Alto, Calif.), and 4064 was supplied by R. Curtiss III (Washington University, St. Louis, Mo.). TABLE 1 Attenuated vaccine strainLB5010 (2) and then transduced by using bacteriophage P22 (Int?) as previously described (37) into BRD175, BRD509, BRD578, BRD726, and SL3261. strains. Female 6- to 8-week-old BALB/c mice were obtained from The University of Melbourne, VCA-2 Department of Microbiology animal facility. For oral immunizations, bacteria were grown for 24 h without shaking before being resuspended in aliquots of PBS Phosphoramidon Disodium Salt (200 l/mouse). Mice were orally immunized via a gastric lavage needle. The dose given to each mouse was determined by retrospective viable count. Thirty minutes prior to oral inoculation, mice were administered 100 l of 10% sodium bicarbonate to neutralize stomach acidity. For antibody and challenge studies, mice were immunized on day 0 and were boosted again on day 28. In colonization studies and in T-cell proliferation and cytokine assays, mice were immunized at day 0 only. Isolation of salmonellae from spleens and Peyers patches of mice. On days 7, 14, and 21 after oral administration of attenuated strains (1 1010 to 3 1010 bacteria per mouse), mice were killed and their spleens and Peyers patches were removed. Spleens and Peyers patches were homogenized in 5 ml of sterile PBS, using a Stomacher 80 (Seward Medical, London, England) homogenizer. The number of salmonellae present in the organs was determined by viable counting of serial dilutions on Luria-Bertani (LB) agar plates containing antibiotic selection for the attenuated strain with and without ampicillin (75 g/ml). Measurement of antibody responses by ELISA. Following oral immunization on day 0 (7 109 to 2 1010 bacteria per mouse), mice were bled weekly from the retro-orbital sinus from days 14 to 56. The titers of antibody present in mouse sera were determined by using a standard enzyme-linked immunosorbent assay (ELISA) or a kinetic ELISA (20, 36). Ninety-six-well Maxisorp immunoplates (Nunc A/S, Kamstrup, Denmark) were coated overnight at 4C with either JM101 and purified by using a polyhistidine affinity tag located at the.