Team:Slovenia/Results/Real-life results





Immunization induced antibody response against CF-multi (left) and CF-UreB (right) proteins in serum after three weeks. Histogram presents mean OD value measured at 450 nm and standard deviation. Mice (n=5) were immunized with CF-multi (Fig. A) and CF-UreB (Fig. B) protein. Sera of non-immunized mice served as negative controls. Sera were collected 3 weeks after first immunization and analyzed by ELISA for IgG antibodies specific to recombinant CF-multi (Fig. A) and UreB (Fig. B) antigen. Recombinant proteins induced significant increase in antigen-specific serum IgG antibodies in comparison to negative control.

Additionally, we tested whether sera of mice immunized with CF-multi contained antibodies specific for ureaseB (Fig.C) and whether sera of mice immunized with CF-UreB (Fig. D) contained antibodies specific for chimeric flagellin. Indeed, ELISA test showed that both, urease B epitope and chimeric flagellin induce serum antibody production as shown in the figures below.

Immunization with flagellin-fused multiepitope produces antibodies that crossreact with whole urease B and vice versa. Histogram presents mean OD value measured at 450 nm and standard deviation. Sera of mice, vaccinated with CF-multi, crossreacted with UreB, (Fig. C) and sera of mice, vaccinated with CF-UreB, crossreacted with CF-multi coating (Fig. D), demonstrating the validity of designed multiepitope approach.

In addition to evaluating antibody production to our recombinant protein vaccines, we also tested whether immunized serum recognizes live Helicobacter pylori bacteria, that would be encountered during infection. To examine this, flow cytometry analysis using goat F(ab)2 anti-mouse IgG-PE was applied and demonstrated that serum IgG indeed interacted with H. pylori. As presented in Fig. A bellow, serum of non-immunized animals did not react with bacteria, whereas serum of animals, immunized with CF-multi recombinant protein recognised bacteria (Fig. C), indicating that opsonizing serum IgG antibodies could induce a cell-mediated process of eradicating the bacterial enemy.



Serum from chimeric flagellin-multiepitope immunized mice reacts with live H. pylori bacteria. Analysis of interactions of serum antibodies with Helicobacter pylori by flow cytometry. H. pylori was harvested from a liquid culture and incubated for 1h with/without serum. After washing, it was incubated for another 1h with secondary goat F(ab)2 anti-mouse IgG-PE and analyzed on a Epics Altra (Beckman-Coulter Electronics) Flow Cytometer Cell Sorter. A: Negative control: Helicobacter pylori incubated with serum of non-immunized animal. B: Secondary antibody specifity control: Helicobacter pylori with secondary antibodies only. C: Helicobacter pylori incubated with serum of immunized animal.

Future studies involving infection of prophylactically vaccinated animal models with H. pylori and evaluation of the decrease in colonization of bacteria in the stomach will further determine how the identified vigorous and early antibody response will translate to eventual therapeutic effect and possibly eradication of the induced infection. On the other hand, we will also examine a therapeutic value of our vaccines by first challenging H. pylori-free mice with these bacteria and evaluate a potential of vaccinations of already infected animals to eradicate H. pylori.