DNA vaccine containing Flagellin A gene induces significant immune responses against Helicobacter pylori infection: An in vivo study

Abstract

Objectives: Helicobacter pylori is one of the most prevalent human infectious agents that is directly involved in various upper digestive tract diseases. Although antibiotics-based therapy and proton pump inhibitors eradicate the bacteria mostly, their effectiveness has been declined recently due to emergence of antibiotic-resistant strains. Development of a DNA vaccine is a promising approach against bacterial pathogens. Genes encoding motility factors are promising immunogens to develop a DNA vaccine against H. pylori infection due to critical role of these genes in bacterial attachment and colonization within the gastric lumen. The present study aimed to synthesize a DNA vaccine construct based on the Flagellin A gene (flaA), the predominant flagellin subunit in H. pylori flagella.

Materials and methods: The coding sequence of flaA was amplified through PCR and sub-cloned in the pBudCE4.1 vector. The recombinant vector was introduced into the human dermal fibroblast cells, and its potency to express the flaA protein was analyzed using SDS-PAGE. The recombinant construct was intramuscularly (IM) injected into the mice, and the profiles of cytokines and immunoglobulins were measured using ELISA.

Results: It has been found that flaA was successfully expressed in cells. Recombinant-vector also increased the serum levels of evaluated cytokines and immunoglobulins in mice.

Conclusion: These findings showed that the pBudCE4.1-flaA construct was able to activate the immune responses. This study is the first step towards synthesis of recombinant-construct based on the flaA gene. Immunization with such construct may inhibit the H. pylori-associated infection; however, further experiments are urgent.

Keywords: DNA vaccine; Flagellin; Helicobacter pylori; Immunomodulation; In vivo; flaA protein.

Figure 1

SDS-PAGE analysis of Helicobacter pylori flaA protein. SDS-PAGE was used to analyze the expression of rflaA. Recombinant vectors pBudCE4.1–flaA and pBudCE4.1 were transfected into HDF. All samples were analyzed by SDS-PAGE, and the protein was stained with Coomassie blue in the gel. Lane SM: molecular mass marker in 10 kDa; Lane 1: whole cell lysate of the HDF with pBudCE4.1; Lane 2: HDF including pBudCE4.1–flaA, 6 hr after transfection; Lanes 3–4, the whole cell lysate of HDF contained recombinant pBudCE4.1–flaA, after 48 hr; Lane 5: Blank control

Figure 2

Increased levels of IgM and IgG following injection of pBudCE4.1-flaA recombinant construct through four booster injections to mice model of experimental

Figure 3

Increased level of IL-2, IL-4, IL-12 and IFN-γ following the injection of pBudCE4.1-flaA recombinant construct through four times booster injections to mice model of experimental