Team:Slovenia/Results/Real-life results

To examine if DNA vaccine constructs were successfully introduced and expressed in the leg muscles upon electroporation, we tagged one of our DNA constructs with GFP gene (multi-TMTIR4-GFP). The part A of the figure below is the image taken under the fluorescent filters demonstrating GFP expression within the leg muscle (arrows) whereas figure B shows the same area under the white light illumination. Part C of the figure represents a positive control. These results confirmed that our electroporation procedure was successful in that the DNA was introduced into the leg muscles and that the expression of our DNA vaccine construct took place in the target tissue. This is a prerequisite for eliciting an immune response to DNA vaccines in vivo. Subsequent to these results, which confirmed the validity of our procedure and expression of constructs in the animal tissue, we electroporated DNA vaccine constructs which will be tested for their prophylactic and therapeutic effects against H. pylori in the near future.

Expression of DNA vaccine coding for multi-TMTIR4-GFP immunogen is detected in vivo in animals using non-invasive optical imaging. DNA vaccine construct (multi-TMTIR4-GFP) tagged with the reporter GFP gene was electroporated into the right leg muscle musculus tibialis cranialis. (A) Transcutaneous image under fluorescent stereoscope – arrows indicate GFP expression. (B) Same area as in part A but the image was taken under the white light illumination. (C) Positive control: leg muscle electroporated with a plasmid coding for an enhanced GFP reporter gene (pEGFP-N1, Clontech laboratories USA) showing areas of GFP expression (arrows).

Animal testing of recombinant protein vaccines

To explore the potential of CF-multi and CF-UreB recombinant protein as an effective vaccine for prophylactic protection of C57BL/6J mice against prospective infection with H. pylori, serum IgG antibody responses were examined by ELISA. Samples were collected in the third week after the first vaccination. CF-multi (Fig. A), UreB (Fig. B) and heat-killed whole cell preparations of H. pylori were used as antigens for coating. Non-immunized mice served as controls. Dilution series and antibody titers of anti-CF-multi protein vaccine are presented in Fig. A below whereas titers of anti-CF-UreB antibodies are shown in Fig. B below. Results unequivocally demonstrate that the prophylactic immunization with both engineered chimeric flagellin recombinant proteins induced a significant increase in antigen-specific serum IgG antibodies already 3 weeks post vaccination suggesting an intense immune response to our vaccines. Moreover, anti-CF-multi and anti-CF-UreB antibodies also reacted with heat-killed H. pylori antigens as well as with living bacteria, which was demonstrated also by flow cytometry (see below). This implies that serum antibodies recognize not only purified recombinant protein molecules (Figs. A and B), but also native epitopes of H. pylori. This result suggests that vaccination with our engineered recombinant proteins should be capable of establishing immune system memory to mobilize relevant immune cells once an animal is challenged with H. pylori infection.

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.