Wen-Tao Yang

Immunoprotection against influenza virus H9N2 by the oral administration of recombinant Lactobacillus plantarumNC8 expressing hemagglutinin in BALB/c mice.

The H9N2 avian influenza virus (AIV) has become increasingly concerning due to its role in severe economic losses in the poultry industry. Transmission of AIV to mammals, including pigs and humans, has accelerated efforts to devise preventive strategies. To develop an effective oral vaccine against H9N2 AIV, a recombinant Lactobacillus plantarum NC8 strain expressing the hemagglutinin (HA) gene of H9N2 AIV was constructed in this study. Mice were orally immunized with the recombinant NC8-pSIP409-HA strain, and sIgA, IgG and HI antibodies were produced by the NC8-pSIP409-HA strain, which also induced CD8(+) T cell immune responses. Most importantly, oral administration produced complete protection against challenge with mouse-adapted H9N2 virus. These results indicate that the recombinant NC8-pSIP409-HA was more effective at inducing the mucosal, humoral and cellular immune responses. Therefore, L. plantarum NC8-pSIP409-HA could become a promising oral vaccine candidate against H9N2 AIV.

Lactobacillus plantarum vaccine vector expressing hemagglutinin provides protection against H9N2 challenge infection.

Hemagglutinin (HA) has been demonstrated as an effective candidate vaccine antigen against AIVs. Dendritic cell-targeting peptide (DCpep) can enhance the robustness of immune responses. The purpose of this study was to evaluate whether DCpep could enhance the immune response against H9N2 AIV when utilizing Lactobacillus plantarum NC8 (NC8) to present HA-DCpep in mouse and chicken models. To accomplish this, a mucosal vaccine of a recombinant NC8 strain expressing HA and DCpep that was constructed in a previous study was employed. Orally administered NC8-pSIP409-HA-DCpep elicited high serum titers of hemagglutination-inhibition (HI) antibodies in mice and also induced robust T cell immune responses in both mouse and chicken models. Orally administered NC8-pSIP409-HA-DCpep elicited high serum titers of hemagglutination-inhibition (HI) antibodies in mice and also induced robust T cell immune responses in both mouse and chicken models. These results revealed that recombinant L. plantarum NC8-pSIP409-HA-DCpep is an effective vaccine candidate against H9N2 AIVs.

Protective efficacy of Fc targeting conserved influenza virus M2e antigen expressed by Lactobacillus plantarum.

Abstract The influenza A (H1N1) virus is a highly contagious acute respiratory disease affecting pigs and humans. This disease causes severe economic loss in many countries, and developing mucosal vaccines is an efficient strategy to control the influenza virus. The neonatal Fc receptor (FcRn) plays an important role in transferring IgG across polarized epithelial cells. In the present study, an oral vaccine was developed using Lactobacillus plantarum to deliver the internal influenza viral protein M2e fused to an IgG Fc fragment. Oral vaccination with recombinant L. plantarum expressing 3M2e‐Fc elicited Peyers patch (PP) DC activation, improved the number of gamma interferon (IFN‐&ggr;)‐producing T cells and increased the frequency of CD8+IFN‐&ggr;+ cells in the mesenteric lymph nodes (MLNs). In addition, the recombinant L. plantarum can induce PP B220+IgA+ expression and enhance specific sIgA secretion and the shaping of growth centers (GCs) in PPs. Furthermore, the data demonstrated that immunization with recombinant L. plantarum expressing 3M2e‐Fc markedly reduced the viral load in the lung and protected against H1N1 influenza virus and mouse‐adapted H9N2 avian influenza virus (AIV) challenge in BALB/c mice. Collectively, the data also showed that this vaccine strategy provided effective protective immunity against infection with homologous and heterologous influenza viruses in a mouse model and may be useful for future influenza vaccine development. HighlightsActivation of CD80 and CD86 molecules on DCs by NC8‐pSIP409‐3M2e‐Fc.IFN‐&ggr;+ producing CD8+ T cells in the MLN were induced by the NC8‐pSIP409‐3M2e‐Fc in mice.Total IgA and IgA+B220+ cells in PP and MLN cells were significantly induced by the NC8‐pSIP409‐3M2e‐Fc in mice.Specific sIgA and activated B cell in the growth center of PP were induced by the NC8‐pSIP409‐3M2e‐Fc in mice.NC8‐pSIP409‐3M2e‐Fc provided 60% and 70% protection against the A/Puerto Rico/8/34(H1N1) and mH9N2 AIV, respectively.

Anti-tumour immune effect of oral administration of Lactobacillus plantarum to CT26 tumour-bearing mice

Colorectal cancer (CRC) is one of the most prevalent forms of cancer that shows a high mortality and increasing incidence. There are numerous successful treatment options for CRC, including surgery, chemotherapy, radiotherapy and immunotherapy; however, their side effects and limitations are considerable. Probiotics may be an effective strategy for preventing and inhibiting tumour growth through stimulation of host innate and adaptive immunity. We investigated and compared potential anti-tumour immune responses induced by two isolated Lactobacillus strains, Lactobacillus plantarum A and Lactobacillus rhamnosus b, by pre-inoculating mice with lactobacilli for 14 days. Subsequently, subcutaneous and orthotopic intestinal tumours were generated in the pre-inoculated mice using CT26 murine adenocarcinoma cells and were assessed for response against the tumour. Our results indicated that oral administration with L. plantarum inhibited CT26 cell growth in BALB/c mice and prolonged the survival time of tumour-bearing mice compared with mice administered L. rhamnosus. L. plantarum produced protective immunity against the challenge with CT26 cells by increasing the effector functions of CD8+ and natural killer (NK) cell infiltration into tumour tissue, up-regulation of IFN-γ (but not IL-4 or IL-17) production, and promotion of Th1-type CD4+ T differentiation. Consequently, our results suggest that L. plantarum can enhance the anti-tumour immune response and delay tumour formation.

Cross-protective efficacy of dendritic cells targeting conserved influenza virus antigen expressed by Lactobacillus plantarum

Avian influenza virus (AIV) can infect birds and mammals, including humans, and are thus a serious threat to public health. Vaccination is vital for controlling AIV circulation. In this study, we generated a recombinant lactobacillus expressing the NP-M1-DCpep of H9N2 avian influenza virus and evaluated the activation effect of NC8-pSIP409-NP-M1-DCpep on dendritic cells (DCs) in a mouse model. The specific mucosal antibody responses and B and T cell responses in lymphoid tissues were also characterized. Importantly, we confirmed that specific CD8 T cells presented in vitro and antigen-specific cytotoxicity (activated the expression of CD107a) and in vivo antigen-specific cytotoxicity after vaccination. The adoptive transfer of NC8-pSIP409-NP-M1-DCpep-primed CD8+ T cells into NOD-SCID mice resulted in effective protection against mouse-adapted AIV infection. In addition, we observed protection in immunized mice challenged with mouse-adapted H9N2 AIV and H1N1 influenza virus, as evidenced by reductions in the lung virus titers, improvements in lung pathology, and weight loss and complete survival. Our data are promising for the generation of effective, non-traditional influenza vaccines against AIVs.

Protection of chickens against H9N2 avian influenza virus challenge with recombinant Lactobacillus plantarum expressing conserved antigens

Avian influenza virus (AIV) is spreading worldwide and is a serious threat to the health of poultry and humans. In many countries, low pathogenic AIVs, such as H9N2, have become an enormous economic burden on the commercial poultry industry because they cause mild respiratory disease and decrease egg production. A recombinant Lactobacillus plantarum NC8 strain expressing NP-M1-DCpep from H9N2 AIV has been studied in a mouse model. However, it remains unknown whether this L. plantarum strain can induce an immune response and provide protection against H9N2 AIV in chickens. In this study, chickens that were orally vaccinated with NC8-pSIP409-NP-M1-DCpep exhibited significantly increased T cell-mediated immune responses and mucosal sIgA and IgG levels, which provided protection against H9N2 AIV challenge. More importantly, compared with oral administration of NC8-pSIP409-NP-M1-DCpep, intranasal administration induced stronger immune responses and provided effective protection against challenge with the H9N2 virus by reducing body weight loss, lung virus titers, and throat pathology. Taken together, these findings suggest that L. plantarum expressing NP-M1-DCpep has potential as a vaccine to combat H9N2 AIV infection.

Construction and immunological evaluation of recombinant Lactobacillus plantarum expressing HN of Newcastle disease virus and DC- targeting peptide fusion protein.

Newcastle disease virus (NDV) has been considered as one of the most severe threats to poultry industry. In this study, we constructed a series of food grade recombinant Lactobacillus plantarum (L. plantarum) strains (RLP) synthesizing either virus hemagglutinin-neuraminidase protein (HN) alone or HN fused with DC cells targeting peptides (DCpep), named RLP(pSIP409-HN) and RLP (pSIP409-HN-DCpep), respectively. The immune responses and protective efficacy were then evaluated in chickens. Results showed that the presence of DCpep in RLP (pSIP409-HN-DCpep) group significantly increased the production of secretory immunoglobulin A (SIgA) in intestines and the percentages of CD3(+)CD4(+) T cells in spleen and peripheral blood leukocytes (P<0.05) compared to chickens immunized with RLP (pSIP409-HN). In addition, the similar enhancement effects were also observed with regard to trachea SIgA, T lymphocytes proliferation and survival rates after NDV challenge, even without significant statics differences. The results demonstrated the possibility to take use of DCpep as an immune adjuvant in the design of NDV vaccine.

β-glucans from Coriolus versicolor protect mice against S. typhimurium challenge by activation of macrophages

The effects of β-glucans from Coriolus versicolor (CVP), which are extracted from a well-known immune stimulator C. versicolor, have been demonstrated extensively in vitro and in vivo. However, until now, the phagocytic activity has not been elucidated. Hence, the objective of the present study was to identify the antibacterial activity of CVP or CVP-treated macrophages by an analysis of cell cytotoxicity, phagocytic activity, intracellular bacterial survival, macrophage activation, production of nitric oxide (NO) and expression of inducible nitric oxide synthase (iNOS) in CVP-treated macrophages using flow cytometry, RT-PCR, a gentamicin protection assay, a Nitric oxide assay and an iNOS enzymatic activity assay. The results indicate that CVP-treated macrophages can phagocytize and kill bacteria, probably due to the production of NO and iNOS. More importantly, CVP-treated macrophages are effective at protecting mice against the challenge of Salmonella typhimurium. The results of this study suggest that the antibacterial effects of CVP are probably caused by the activation of innate immune cells, especially macrophages, because the activated macrophage produces NO, which kills bacteria. These phenomena indicate the possibility of CVP as a potential alternative for antibiotics against resistant bacteria.

Construction and immunological evaluation of recombinant Lactobacillus plantarum expressing SO7 of Eimeria tenella fusion DC-targeting peptide

The coccidiosis caused by Eimeria tenella (coccidian) and other species is a serious parasitic disease that affects the global poultry breeding industry. Lactobacillus strains exhibit a number of properties that make them attractive candidates as delivery vehicles for presentation to the mucosa of compounds with pharmaceutical interest, particularly vaccines. Here, the recombinant Lactobacillus plantarum (co-expressing SO7 and DCpep gene) was constructed, and its efficacy against E. tenella challenge was evaluated in this study. Broiler chickens were orally immunized with live recombinant L. plantarum NC8-pSIP409-SO7-DCpep for two weeks and were then challenged with 5×104E.tenella sporulated oocysts per chicken. During the experiment, body weight gains, cecum lesion scores, fecal oocyst shedding and antibody responses in serum and intestinal washes were assessed as measures of protective immunity. The results indicated that chickens immunized with live recombinant L. plantarum can increase body weight gains and serum antibody responses compared to the control groups. Meanwhile, fecal oocyst shedding in the immunized group was significantly reduced (p<0.01). Moreover, recombinant L. plantarum can significantly relieve pathological damage in cecum, according to lesion scores and histopathologic cecum sections (p<0.01). Therefore, these results indicate that recombinant L. plantarum NC8-pSIP409-SO7-DCpep could become a promising oral vaccine candidate against E. tenella infection.

Immunogenicity of recombinant Lactobacillus plantarum NC8 expressing goose parvovirus VP2 gene in BALB/c mice.

Goose parvovirus (GPV) continues to be a threat to goose farms and has significant economic effects on the production of geese. Current commercially available vaccines only rarely prevent GPV infection. In our study, Lactobacillus (L.) plantarum NC8 was selected as a vector to express the VP2 gene of GPV, and recombinant L. plantarum pSIP409-VP2/NC8 was successfully constructed. The molecular weight of the expressed recombinant protein was approximately 70 kDa. Mice were immunized with a 2 × 109 colony-forming unit/200 µL dose of the recombinant L. plantarum strain, and the ratios and numbers of CD11c+, CD3+CD4+, CD3+CD8+, and interferon gamma- and tumor necrosis factor alpha-expressing spleen lymphocytes in the pSIP409-VP2/NC8 group were higher than those in the control groups. In addition, we assessed the capacity of L. plantarum SIP409-VP2/NC8 to induce secretory IgA production. We conclude that administered pSIP409-VP2/NC8 leads to relatively extensive cellular responses. This study provides information on GPV infection and offers a clear framework of options available for GPV control strategies.