Anti-Helicobacter pylori activity and gastroprotective effects of human stomach-derived Lactobacillus paragasseri strain LPG-9.

The eradication of Helicobacter pylori is an urgent global issue. However, the traditional regimens have several limitations. Thus, we propose the idea of treating bacterial gastric disease with the objective of eliminating gastric pathogenic bacteria and enhancing gastroprotective effects using gastric probiotics. In this study, a total of 12 Lactobacillus strains were isolated from the gastric mucosa of healthy donors. After evaluation using a weight scoring system, Lactobacillus paragasseri strain LPG-9 was identified as the most promising probiotic for gastric disease, with the highest acid-resistance and the best adhesion characteristics. Gastric colonisation, H. pylori inhibition, anti-inflammatory, and gastric homeostasis effects of LPG-9 were confirmed in C57BL/6 mice. Finally, a safety evaluation and whole-genome sequencing were performed. Based on the results of this study, LPG-9 originates from the gastric microbiota and is a promising probiotic for gastric disease, particularly H. pylori-induced gastritis, providing a solution to this global issue.

Oral immunization of mice using Bifidobacterium longum expressing VP1 protein from enterovirus 71.

Bifidobacterium longum is an attractive candidate for delivering biologically active proteins by the mucosal route due to its non-pathogenic and colonizing properties. Enterovirus 71 (EV71) has aroused widespread attention recently due to several epidemics, and great attention should be paid to the fact that there are currently no effective antiviral drugs or vaccines against EV71 infection. In this report, we described a recombinant B. longum that could be used to develop an oral vaccine against EV71 infection. A VP1 expression vector (pBBADs-VP1) was constructed by amplifying the EV71 VP1 gene and inserting it into the E. coli-Bifidobacterium shuttle expression vector pBBAD/Xs. Then, the expression of VP1 protein in pBBADs-VP1-transformed bacteria was demonstrated by western blot. In vivo studies indicated that oral immunization of BALB/c mice with pBBADs-VP1-transformed bacteria induced potent immune responses against EV71 infection, including virus-neutralising titers, anti-EV71-VP1 antibody and the induction of Th1 immune responses in the spleen and Peyer's patches. Importantly, immunization of mother mice with this recombinant VP1-expressing B. longum conferred protection to neonatal mice. These results demonstrate that the novel oral vaccine utilizing B. longum expressing the VP1 protein might successfully elicit a specific immune response against EV71 infection.

Bifidobacterium as an oral delivery carrier of interleukin-12 for the treatment of Coxsackie virus B3-induced myocarditis in the Balb/c mice.

IL-12 plays an important role in the treatment of many infectious diseases by being administered intravenously or intramuscularly. However, intravenous or intramuscular administration is difficult and inconvenient and may cause side effects. The aim of this study is to develop a novel oral delivery system for IL-12 using genetically engineered Bifidobacterium longum as the carrier and further investigate the efficacy of IL-12-expressed B. longum on the coxsackie virus B3 (CVB3)-induced myocarditis in mice. A mIL-12 gene expression vector pBBADs-IL-12 for B. longum was constructed and transformed into Bifidobacterium. Subsequently, the expression of mIL-12 in the engineered B. longum was identified in vitro by western blot and enzyme-linked immunosorbent assay (ELISA) after l-arabinose induction. Moreover, our data indicated that oral administration of IL-12-expressed B. longum for two weeks after CVB3 infection in the Balb/c mice could downregulate the severity of virus-induced myocarditis, markedly reduce the virus titers in the heart and induce a Th1 pattern in the spleen and heart compared with the controls. In conclusion, a novel oral delivery system of Bifidobacterium for murine IL-12 has been successfully established. Oral administration of mIL-12-transformed B. longum may play a therapeutic role in the treatment of CVB3-induced myocarditis in the mice.

Oral administration of interferon-α2b-transformed Bifidobacterium longum protects BALB/c mice against coxsackievirus B3-induced myocarditis.

Multiple reports have claimed that low-dose orally administered interferon (IFN)-α is beneficial in the treatment of many infectious diseases and provides a viable alternative to high-dose intramuscular treatment. However, research is needed on how to express IFN stably in the gut. Bifidobacterium may be a suitable carrier for human gene expression and secretion in the intestinal tract for the treatment of gastrointestinal diseases. We reported previously that Bifidobacterium longum can be used as a novel oral delivery of IFN-α. IFN-transformed B. longum can exert an immunostimulatory role in mice; however the answer to whether this recombinant B. longum can be used to treat virus infection still remains elusive. Here, we investigated the efficacy of IFN-transformed B. longum administered orally on coxsackie virus B3 (CVB3)-induced myocarditis in BALB/c mice. Our data indicated that oral administration of IFN-transformed B. longum for 2 weeks after virus infection reduced significantly the severity of virus-induced myocarditis, markedly down regulated virus titers in the heart, and induced a T helper 1 cell pattern in the spleen and heart compared with controls. Oral administration of the IFN-transformed B. longum, therefore, may play a potential role in the treatment of CVB3-induced myocarditis.

Increased mRNA expression of interferon-induced Mx1 and immunomodulation following oral administration of IFN-alpha2b-transformed B. longum to mice.

We previously constructed an arabinose-inducible recombinant Bifidobacterium longum that could efficiently express secreted IFN-alpha2b in vitro (Deng et al. in Arch Microbiol 191:681-686, 2009). Here, we investigated the influence of oral pBAD-SPIFN-transformed B. longum on immunomodulation and IFN-induced Mx1 gene transcription in mice. We observed enhanced serum and fecal IFN-alpha2b concentrations in mice orally administered recombinant B. longum, suggesting a possible Th1 pattern of induction in the spleen and Peyer's patches. Transcription of the typically IFN-induced antiviral Mx1 gene in the hepatic and intestinal tissues of these mice was also markedly enhanced. In conclusion, oral administration of the recombinant B. longum expressing IFN-alpha2b might play its roles in the immunomodulation of the mice, and the potential clinical value of this bacterium in the treatment of viral infections needs to be further studied.

Signal peptide of Arabinosidase enhances secretion of interferon-alpha2b protein by Bifidobacteria longum.

Bifidobacteria can potentially be used for gene therapy. Here, we reported that 65% of the total hIFN-alpha2b produced from Bifidobacteria longum transformed with pBAD-SPIFN plasmids encoding a fusion protein of the arabinosidase signal peptide and human IFN-alpha2b (hIFN-alpha2b), was secreted. For B. longum transformed with pBAD-IFN plasmids (hIFN-alpha2b without the signal peptide), only 15% of the total IFN-alpha2b was secreted and western blotting and N-terminal amino-acid sequence analysis revealed cleavage of the arabinosidase signal peptide from the secreted hIFN-alpha2b. Moreover, the active level of the secreted hIFN-alpha2b in the supernatant of B. longum transformed with pBAD-SPIFN plasmids was over 1,000 IU/ml commercial rhIFN-alpha2b. Hence, the arabinosidase signal peptide can enhance the secretion efficiency of IFN-alpha2b from B. longum.