Oral administration of DNA alginate nanovaccine induced immune-protection against Helicobacter pylori in Balb/C mice

Abstract

Background: Helicobacter pylori (H. Pylori), is an established causative factor for the development of gastric cancer and the induction of persistent stomach infections that may lead to peptic ulcers. In recent decades, several endeavours have been undertaken to develop a vaccine for H. pylori, although none have advanced to the clinical phase. The development of a successful H. pylori vaccine is hindered by particular challenges, such as the absence of secure mucosal vaccines to enhance local immune responses, the absence of identified antigens that are effective in vaccinations, and the absence of recognized indicators of protection.

Methods: The DNA vaccine was chemically cloned, and the cloning was verified using PCR and restriction enzyme digestion. The efficacy of the vaccination was investigated. The immunogenicity and immune-protective efficacy of the vaccination were assessed in BALB/c mice. This study demonstrated that administering a preventive Alginate/pCI-neo-UreH Nanovaccine directly into the stomach effectively triggered a robust immune response to protect against H. pylori infection in mice.

Results: The level of immune protection achieved with this nano vaccine was similar to that observed when using the widely accepted formalin-killed H. pylori Hel 305 as a positive control. The Alginate/pCI-neo-UreH Nanovaccine composition elicited significant mucosal and systemic antigen-specific antibody responses and strong intestinal and systemic Th1 responses. Moreover, the activation of IL-17R signaling is necessary for the defensive Th1 immune responses in the intestines triggered by Alginate/pCI-neo-UreH.

Conclusion: Alginate/pCI-neo-UreH is a potential Nanovaccine for use in an oral vaccine versus H. pylori infection, according to our findings.

Keywords: Alginate, UreH gene; Helicobacter pylori; Nanovaccine.

Fig. 1

(A) The UreH gene was inserted into the pCI-neo expression vector using XhoI and NotI. (B) The effective synthesis of the recombinant plasmid was verified using electrophoretic extraction of the UreH digestion fragments. 1: pCI-neo-UreH recombinant plasmid before digestion, 2: pCI-neo-UreH recombinant plasmid after digestion, M: Marker III DNA Ladder. (C) Co-transfection of the recombinant constructs to the HEK 293-T cells resulted in 810 bp bands after PCR analyses using specific primers. 1: Negative control, 2: 810 bp bands of UreH gene, M: Marker III DNA Ladder. (D) The number of NPs in each interaction determines the relative fluorescence of pDNA in each sample. Free-pCI-neo has the most fluorescence. Fluorescence intensities decrease in the Alginate/pCI-neo-UreH. (E) Nanoparticle size measurement following storage by freezing at − 20 °C for 1 month. (F) Electrophoretic movement of different formulations of nanovaccines. Lane M: negative control; Lane 1: Free-pCI-neo; Lane 2: Alginate/pCI-neo-UreH; Lane 3: Alginate/pCI-neo. (G) In vitro release kinetic of different formulations of nano vaccines at pH 7. (H) Cytotoxicity of several nano vaccine formulations on HEK-293 cells after 24 h (n = 3; mean ± standard error; ns: not significant; *p ≤ 0.05; **p ≤ 0.01, ***p ≤ 0.001)

Fig. 2

(A) Transcription of the recombinant vaccine at mRNA level. L1: Negative control; L2 and L3: Alginate/pCI-neo-UreH; L4 and L5: pCI-neo-UreH; M: 100 bp DNA marker. (B) Expression of recombinant Alginate/pCI-neo-UreH in protein level. L1: Negative control; L2: Alginate/pCI-neo; L3: Alginate/pCI-neo-UreH; L4: pCI-neo-UreH; M: protein marker. (C) Bacterial burdens in the stomach tissues of BALB/c mice 5 days’ post-challenge. The stars (*) are according to the PBS group. IgG (D), IFN-γ (E) and IL-17 A(F) levels were found in the splenocyte supernatants of the control and immunized groups. PBS was used as a control. * p < 0.05, ** p < 0.01, *** p < 0.001

Fig. 3

Th1 responses are specific to the H. pylori antigen are selectively boosted by oral vaccination with Alginate/pCI-neo-UreH and Orally immunized mice with whole-cell killed H. pylori Hel 305 and other groups. Two weeks after the previous immunization cycle, cells were extracted from the spleen (A, C) and mesenteric lymph nodes (B, D). ELISA collected and analysed Supernatants for either IFN-γ (A, B) or IL-17 A (C, D). Data from three trials with n = 5 mice per group and experiment. **p < 0.01, *** < 0.001

Fig. 4

Increased intestine IgA responses to the H. pylori antigen by alginate/pCI-neo-UreH. Vaccines were Orally administered to mice. ELISA measured specific IgA antibody titers in fecal pellet supernatants before each immunization cycle and two weeks after the final immunization round. A) For each group of mice that received the vaccine, the graph line depicts the change in IgA titers over time. Animals were infused to extract the blood from the organs two weeks after the second immunization round, and tissue was recovered. IgA titers specific to H. pylori were assessed in extracts from the (B) stomach, (C) jejunum, and (D) ileum. Data from three separate trials with an average of five mice per group and experiment. *p < 0.05, **p < 0.01, ***p < 0.00

Fig. 5

Alginate/pCI-neo-UreH vaccinations were administered intragastrically to WT, IL-17R-/-mice to elicit intestinal, but not systemic, Th1-type responses. Two weeks after the prior vaccination, cells were isolated from the spleen and mesenteric lymph nodes (MLN) and restimulated ex vivo with purified MP305 for 72 h. Supernatants were obtained and subjected to an IgA test from the fecal pellet extract (A), jejunum (B), ileum (C), and colon (D). Results from two distinct investigations, each comprising five animals in each group. *p < 0.05, **p < 0.01; ***p < 0.001