Pilot study of phoP/phoQ-deleted Salmonella enterica serovar typhimurium expressing Helicobacter pylori urease in adult volunteers

Abstract

Attenuated Salmonella enterica serovar Typhi has been studied as an oral vaccine vector. Despite success with attenuated S. enterica serovar Typhimurium vectors in animals, early clinical trials of S. enterica serovar Typhi expressing heterologous antigens have shown that few subjects have detectable immune responses to vectored antigens. A previous clinical study of phoP/phoQ-deleted S. enterica serovar Typhi expressing Helicobacter pylori urease from a multicopy plasmid showed that none of eight subjects had detectable immune responses to the vectored antigen. In an attempt to further define the variables important for engendering immune responses to vectored antigens in humans, six volunteers were inoculated with 5 x 10(7) to 8 x 10(7) CFU of phoP/phoQ-deleted S. enterica serovar Typhimurium expressing the same antigen. Two of the six volunteers had fever; none had diarrhea, bacteremia, or other serious side effects. The volunteers were more durably colonized than in previous studies of phoP/phoQ-deleted S. enterica serovar Typhi. Five of the six volunteers seroconverted to S. enterica serovar Typhimurium antigens and had strong evidence of anti-Salmonella mucosal immune responses by enzyme-linked immunospot studies. Three of six (three of five who seroconverted to Salmonella) had immune responses in the most sensitive assay of urease-specific immunoglobulin production by blood mononuclear cells in vitro. One of these had a fourfold or greater increase in end-point immunoglobulin titer in serum versus urease. Attenuated S. enterica serovar Typhimurium appears to be more effective than S. enterica serovar Typhi for engendering immune responses to urease. Data suggest that this may be related to a greater stability of antigen-expressing plasmid in S. enterica serovar Typhimurium and/or prolonged intestinal colonization. Specific factors unique to nontyphoidal salmonellae may also be important for stimulation of the gastrointestinal immune system.

FIG. 1

Chromosomal deletion and plasmid map for S. enterica serovar Typhimurium LH1160. The chromosomal deletion (ΔphoP ΔphoQ ΔpurB) between endogenous NcoI sites is shown. The purB and phoP genes are contiguous. The urease expression plasmid encodes the urease A and B subunit genes, ureA and ureB (without other regulatory or structural elements of urease), cloned behind the chloramphenicol acyltransferase promoter (Pcat). The purB gene encodes adenylosuccinate lyase enzyme, which catalyzes an essential step in the de novo synthesis of AMP, was cloned from S. enterica serovar Typhimurium, and is driven from its native promoter. The origin of replication (ori) of the plasmid is derived from plasmid pBR328, a moderate-copy-number plasmid.

FIG. 2

Cell-associated urease A and B within attenuated phoP/phoQ-deleted Salmonella vectors. Bacterial strains were grown to early stationary phase (12 h), as for clinical studies. Whole-bacterial-cell protein lysates were made, separated by SDS-PAGE, and either stained with Coomassie blue (left panel) or blotted to nitrocellulose and probed with a polyclonal antibody for H. pylori urease (right panel). S. enterica serovar Typhi and Typhimurium strains carrying either the urease-expressing stabilized plasmid (+) or the isogenic “empty plasmid” lacking the ureA and ureB genes (−) are shown side by side. Loading was normalized by CFU determinations such that each lane contains protein from 5 × 10⁷ CFU. As a control, 1 μg of recombinant urease A and B was loaded (lane C). The immunoblot was developed with a chemiluminescent substrate, and an autoradiogram is shown. Urease A and B subunits (arrows) are easily visualized on both the Coomassie-stained gel and immunoblot, and the amounts are indistinguishable between serotypes.

FIG. 3

In vitro stability of stabilized plasmids in S. enterica serovar Typhi and Typhimurium strains. Bacterial strains were grown as for the generation of volunteer inocula in Luria broth. Aliquots were removed at the designated times and plated in duplicate for CFU determinations and assessment of colonies size. The percentage of large-morphology colonies (those which retained purB-bearing plasmids) is shown. The S. enterica serovar Typhimurium LH1160 tested here is denoted by solid triangles, and the previously evaluated S. enterica serovar Typhi 1033 is denoted by solid squares. Empty plasmid controls lacking the ureA and ureB genes are denoted by open symbols. The data suggest that more rapid loss of plasmid in vitro is related to the presence of the ureA and ureB genes in S. enterica serovar Typhi, but not in S. enterica serovar Typhimurium.

FIG. 4

Assay of vaccine-specific IgG produced by mononuclear cells in vitro. Mononuclear cells were isolated on the study day noted and cultured for 48 h. Culture supernatants were applied in duplicate to ELISA plates coated with either recombinant urease (A) or S. enterica serovar Typhimurium LPS (B) or flagella (C). The plates were developed with a peroxidase-labelled goat anti-human antibody directed against human IgG. A greater than threefold increase in optical density over the baseline day 0 value was considered a positive result. Samples from volunteers who had a threefold or greater increase in optical density in the urease-contained wells are denoted by solid symbols and solid lines (volunteers 1, 3, and 6). Samples from volunteers who did not have an increase IgG directed against urease are represented by open symbols and dotted lines. All volunteers except volunteer 2 had much larger increases in optical density in LPS and flagellum wells, and the y-axis scales differ for the panels, reflecting this finding. PBMC isolated from healthy, unimmunized H. pylori-seronegative volunteers had “flat” profiles similar to those of volunteer 2 (data not plotted).