Recombinant Lactobacillus plantarum NC8 strain expressing porcine rotavirus VP7 induces specific antibodies in BALB/c mice.

The major etiologic agent that causes acute gastroenteritis worldwide in young animals and children is Group A rotavirus. Currently, commercially available vaccines do not often prevent porcine rotavirus (PRV) infection. In this study, we evaluated the efficacy of oral recombinant Lactobacillus vaccine against PRV in a mouse model. Lactobacillus plantarum NC8 was used as the host strain, and bacterial vectors were constructed, because the NC8 isolated has shown the capability to survive gastric transit and to colonize the intestinal tract of humans and other mammals. To explore the immunological mechanisms, lactic acid bacterial vectors were used to express VP7 antigen from PRV. We constructed an L. plantarum strain with surface-displayed VP7, named NC8-pSIP409-pgsA-VP7-DCpep. The expressed recombinant protein had a molecular weight of ∼37 kDa. The strain was used to immunize BALB/c mice to evaluate their immunomodulatory characteristics. Mice were orally immunized with recombinant L. plantarum NC8-pSIP409-pgsA-VP7-DCpep at a dose of 2 × 109 colony forming units/200 µl. The results showed that NC8-pSIP409-pgsA-VP7-DCpep significantly stimulated the differentiation of dendritic cells (DCs) in Peyer's patches (PPs) and increased the serum levels of IL-4 and IFN-γ, as measured by enzyme-linked immunosorbent assay in mice treated with NC8-pSIP409-pgsA-VP7-DCpep. Compared to the empty vector group, NC8-pSIP409-pgsA-VP7-DCpep significantly increased the production of B220+ B cells in mesenteric lymph nodes (MLNs) and PPs and also increased the titer levels of the VP7-specific antibodies, including IgG and sIgA. The administration of NC8-pSIP409-pgsA-VP7-DCpep mediated relatively broad cellular responses. This study reveals that clear alternatives exist for PRV control strategies and provides information on PRV infection.

Recombinant Lactobacillus plantarum expressing HA2 antigen elicits protective immunity against H9N2 avian influenza virus in chickens.

Low pathogenic H9N2 subtype avian influenza virus (AIV) can lead to moderate respiratory symptoms and low egg production rates in poultry. Due to its immunologic suppression, other various infectious pathogens give rise to the co-infection of hosts, causing heavy economic losses in the commercial poultry industry in both China and worldwide. Therefore, it is time to explore a novel, safe, and effective vaccine. We have already made use of the surface of Lactobacillus plantarum to display AIV HA2 (NC8-pSIP409-pgsA'-HA2), which demonstrated that it has a good immunogenicity. In this study, by evaluating the immune protection effect of NC8-pSIP409-pgsA'-HA2 on chickens, we found that the hemagglutination inhibition (HI) antibodies, specificity IgG antibody in chickens, the sIgA titer in broncho alveolar lavage fluids (BALF), and the T cell response were increased notably after oral vaccination with NC8-pSIP409-pgsA'-HA2. In addition, weight loss, lung titers, and lung pathologies were improved when chickens were orally vaccinated with NC8-pSIP409-pgsA'-HA2 after challenge with H9N2 AIV. This strategy lays the foundation for the development of recombinant L. plantarum oral vaccines in the prevention of AIV.

Expression and purification of swine RAG2 in E. coli for production of porcine RAG2 polyclonal antibodies.

Recombination activating gene 2 (RAG2) is necessary for immature B cell differentiation. Antibodies to human and rabbit RAG2 are currently commercially available, but antibodies to swine RAG remain unavailable to date. In this study, the swine RAG2 genes sequence was synthesized and then cloned into a pET-28a vector. The recombinant fusion protein was successfully expressed in E. coli, purified through nickel column chromatography, and further digested with Tobacco Etch Virus protease. The cleaved protein was purified by molecular-exclusion chromatography and named pRAG2. We used pRAG2 to immunize rabbits, collected the serum and purified rabbit anti-pRAG2 polyclonal antibodies. The rabbit anti-pRAG2 polyclonal antibodies were tested via immunofluorescence on eukaryotic cells overexpressing pRAG2 and also able to recognize pig natural RAG2 and human RAG2 protein in western blotting. These results indicated that the prepared rabbit anti-pRAG2 polyclonal antibodies may serve as a tool to detect immature B cell differentiation of swine.

The regulatory effect of Lactobacillus rhamnosus GG on T lymphocyte and the development of intestinal villi in piglets of different periods.

The maturation and development of T cells were not completed until T cells were selected in thymus. It was not until the early 1960s that j.f.a.p. discovered the importance of thymus in T cell development. Twelve healthy piglets were randomly divided into two groups, the experimental group (LGG group) and the control group (saline group). The LGG group piglets were given 1 ml LGG (6 × 109 CFU/ml) per day. The saline group was given 1 ml of normal saline per day. The piglets were slaughtered at 30 days and 45 days, respectively, and the MLN, jejunum and ileum PPs, LP of the piglets were taken. The expression of CD3+CD4+ T lymphocytes was detected by flow cytometry, and intestinal villi development was observed by intestinal paraffin section. The results showed that the flow cytometry results at 30 days and 45 days showed that the CD3+CD4+ T lymphocytes in the MLN group were significantly different from those in the saline group (P < 0.05, P < 0.01).The CD3+CD4+ T lymphocytes in the jejunum PP of piglets in LGG group were significantly different from those in saline group (P < 0.05). The CD3+CD4+ T lymphocytes in the ileum PP of the LGG group were significantly different from those in the saline group (P < 0.05, P < 0.01). CD3+CD4+ T lymphocytes and normal saline in the piglets of the LGG group There was a significant difference between the two groups (P < 0.001, P < 0.05). P < 0.001). HE staining results showed the length of the LGG group ileal villi in piglets at 30 days, 45 days was significantly different from that in normal saline group (P < 0.01, P < 0.01). LGG can also regulate the proliferation of T lymphocytes in the intestine of early weaned piglets at 30 days and 45 days increase the number of CD3+CD4+ T lymphocytes.

Immune responses of mice inoculated with recombinant Lactobacillus plantarum NC8 expressing the fusion gene HA2 and 3M2e of the influenza virus and protection against different subtypes of influenza virus.

To evaluate the efficiency of preventing pathogenic avian influenza by vaccination with recombinant Lactobacillus plantarum (L. plantarum) that expresses conserved antigens, the mucosal and systemic immune responses in animals vaccinated with recombinant L. plantarum NC8-409-1 (NC8-pSIP409-pgsA'-HA2) and NC8-409-2 (NC8-pSIP409-pgsA'-3M2e-HA2) were evaluated. Our results showed that recombinant L. plantarum NC8-409-1 and NC8-409-2 could substantially stimulate the specific IgA titer in the intestine and the specific IgG antibody titer in the serum. We also found that recombinant L. plantarum induced increases in the number of B220+ IgA+ cells in Peyer's patches (PPs), in lymphocyte proliferation and in the number of IFN-γ+ producing CD8+ T cells after immunization in mice. Most importantly, the mice that were vaccinated with recombinant L. plantarum NC8-409-2 and NC8-409-1 were to some extent protected against infection challenge with the H9N2 and H1N1 viruses. In particular, NC8-409-2 provided up to 80% protection against the H9N2 virus, and NC8-409-1 provided up to 60% protection. Lung tissue pathology was also reduced. Therefore, recombinant L. plantarum NC8-409-2 and NC8-409-1 may be candidate oral vaccines against bird flu.