Harry Kleanthous

Attenuated Salmonella enterica Serovar Typhi Expressing Urease Effectively Immunizes Mice against Helicobacter pylori Challenge as Part of a Heterologous Mucosal Priming-Parenteral Boosting Vaccination Regimen

ABSTRACT Recombinant vaccine strains of Salmonella enterica serovar Typhi capable of expressing Helicobacter pylori urease were generated by transforming strains CVD908 and CVD908-htrA with a plasmid harboring the ureAB genes under the control of an in vivo-inducible promoter. The plasmid did not interfere with the ability of either strain to replicate and persist in human monocytic cells or with their transient colonization of mouse lungs. When administered to mice intranasally, both recombinant strains elicited antiurease immune responses skewed towards a Th1 phenotype. Vaccinated mice exhibited strong immunoglobulin G2a (IgG2a)-biased antiurease antibody responses as well as splenocyte populations capable of proliferation and gamma interferon (IFNγ) secretion in response to urease stimulation. Boosting of mice with subcutaneous injection of urease plus alum enhanced immune responses and led them to a more balanced Th1/Th2 phenotype. Following parenteral boost, IgG1 and IgG2a antiurease antibody titers were raised significantly, and strong urease-specific splenocyte proliferative responses, accompanied by IFNγ as well as interleukin-4 (IL-4), IL-5, and IL-10 secretion, were detected. Neither immunization with urease-expressing S. enterica serovar Typhi alone nor immunization with urease plus alum alone conferred protection against challenge with a mouse-adapted strain of H. pylori; however, a vaccination protocol combining both immunization regimens was protective. This is the first report of effective vaccination against H. pylori with a combined mucosal prime-parenteral boost regimen in which serovar Typhi vaccine strains are used as antigen carriers. The significance of these findings with regard to development of a human vaccine against H. pylori and modulation of immune responses by heterologous prime-boost immunization regimens is discussed.

Immunization of rhesus monkeys with a mucosal prime, parenteral boost strategy protects against infection with Helicobacter pylori

Rhesus monkeys were immunized with recombinant Helicobacter pylori urease vaccine given solely by the parenteral route or preceded by a priming dose given by the oral route. Two groups of monkeys received parenteral urease with either a synthetic glycolipid adjuvant (Bay) or aluminum hydroxide (alum) as adjuvants. A third group of monkeys received a priming dose of oral urease given with the mucosal adjuvant LT (Escherichia coli heat labile enterotoxin), followed by parenterally administered booster doses of urease adsorbed to alum. Monkeys receiving placebo served as controls. The monkeys received a total of 4 doses of vaccine with the first 3 doses given every 3 weeks and the last booster dose administered 14 weeks later. The monkeys were challenged orally with H. pylori one week after the last vaccine dose and euthanized 10 weeks after challenge, at which time, their stomachs were collected for determination of bacterial colonization and histopathology. Monkeys primed with the oral vaccine and boosted with the parenteral vaccine showed a statistically significant reduction in bacterial colonization when compared to sham-immunized control animals (P = 0.05; Wilcoxon rank sums test). Monkeys receiving parenteral only regimes of urease plus Bay or alum showed no difference in bacterial colonization compared with sham-immunized controls (P = 1.00 and P = 0.33, respectively). The mucosal prime-parenteral boost regime did not cause gastropathy. There was no difference in any of the 3 treatment groups with respect to gastric epithelial changes compared to control animals. There was also no difference in the type and extent of gastric inflammatory cell infiltrates between animals vaccinated by the mucosal prime-parenteral boost strategy and sham immunized controls. However, monkeys receiving the two parenteral-only regimens had slightly elevated gastritis scores.

Sterilizing immunity against experimental Helicobacter pylori infection is challenge-strain dependent.

The development of a murine model of Helicobacter pylori infection through serial in vivo passage of candidate strains has enabled a quantitative assessment of vaccine efficacy. In this study we compare infection with and protection against challenge from both CagA(+) type I, and CagA(-) type II in vivo adapted isolates. In vivo passage of a type II H. pylori isolate resulted in a highly infectious strain (X47-2AL), capable of reproducibly infecting mice to high density (10(7) CFU/g of gastric tissue). Similarly adapted type I strains were found to colonize mice at a significantly lower level (10(4)-10(5) CFU/g tissue). Mucosal immunization with recombinant urease (rUre) significantly protected animals against both types. Protection against X47-2AL was characterized by a > or =100-fold (or 2 log) reduction in bacterial density. However, the presence of a residual infection highlighted the inability to achieve sterilizing immunity against this strain. The level of protection appeared independent of challenge dose, and was stable for up to 6 months, all animals exhibiting a low-level residual infection that did not recrudesce with time. Similarly immunized mice challenged with isolates representing the residual infection were also protected, confirming that they did not represent a sub-population of H. pylori that could escape immunity. Immunization and challenge studies with type I adapted-isolates, demonstrated a similar 2-3 log reduction in the bacterial burden, but that in this instance resulted in sterilizing immunity. These results suggest varied specificity for the murine host by different Helicobacter strains that can influence the outcome of both infection and immunity.

Specific Immunoglobulin A Antibodies in Maternal Milk and Delayed Helicobacter pylori Colonization in Gambian Infants

BACKGROUND Immunoglobulin A (IgA) in maternal milk may protect Gambian infants from early Helicobacter pylori colonization. This study sought evidence that this protection could be due to specific IgA antibodies. METHODS Sixty-five infants were screened from 12 weeks of age with [13C]-urea breath tests. Antibodies in maternal milk were measured to determine total IgA content and to detect specific IgA antibodies against crude whole-cell and recombinant H. pylori urease antigen preparations. RESULTS Ten children (15%) had no evidence of early H. pylori colonization, 10 (15%) had early H. pylori colonization, and 43 (66%) had mixed results. Levels of maternal circulating specific immunoglobulin G, total milk IgA, and IgA directed against crude whole-cell H. pylori antigen preparation were not significantly associated with the rate of infant H. pylori colonization. However, mothers of infants with no evidence of early colonization produced significantly higher levels of anti-recombinant urease IgA antibodies in milk than did control mothers, particularly at 8, 16, and 20 weeks postpartum (P<.01). CONCLUSIONS These observations support the hypothesis that antibodies in mothers milk directed against H. pylori urease can protect against colonization in human infancy.