Chunfeng Wang

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.

Reassortant between human-like H3N2 and avian H5 subtype influenza A viruses in pigs: a potential public health risk.

Background Human-like H3N2 influenza viruses have repeatedly been transmitted to domestic pigs in different regions of the world, but it is still uncertain whether any of these variants could become established in pig populations. The fact that different subtypes of influenza viruses have been detected in pigs makes them an ideal candidate for the genesis of a possible reassortant virus with both human and avian origins. However, the determination of whether pigs can act as a “mixing vessel” for a possible future pandemic virus is still pending an answer. This prompted us to gather the epidemiological information and investigate the genetic evolution of swine influenza viruses in Jilin, China. Methods Nasopharyngeal swabs were collected from pigs with respiratory illness in Jilin province, China from July 2007 to October 2008. All samples were screened for influenza A viruses. Three H3N2 swine influenza virus isolates were analyzed genetically and phylogenetically. Results Influenza surveillance of pigs in Jilin province, China revealed that H3N2 influenza viruses were regularly detected from domestic pigs during 2007 to 2008. Phylogenetic analysis revealed that two distinguishable groups of H3N2 influenza viruses were present in pigs: the wholly contemporary human-like H3N2 viruses (represented by the Moscow/10/99-like sublineage) and double-reassortant viruses containing genes from contemporary human H3N2 viruses and avian H5 viruses, both co-circulating in pig populations. Conclusions The present study reports for the first time the coexistence of wholly human-like H3N2 viruses and double-reassortant viruses that have emerged in pigs in Jilin, China. It provides updated information on the role of pigs in interspecies transmission and genetic reassortment of influenza viruses.

Development of a reverse genetics system based on RNA polymerase II for Newcastle disease virus genotype VII

Abstract Newcastle disease virus (NDV) has only a single serotype but diversified genotypes. Genotype VII strains are the prevalent currently circulating genotype worldwide, and in particular, these strains cause outbreaks in waterfowl. In this study, a reverse genetics system for highly virulent NDV isolated from goose flocks was developed independent of conventional T7 RNA polymerase. Infectious virus was successfully generated by an RNA polymerase II promoter to drive transcription of the full-length virus antigenome. A green fluorescent protein (GFP)-expressing virus was generated by inserting an additional transcription cassette coding for the enhanced GFP between the P and M genes of the genome. The expression of GFP was confirmed by western blotting and fluorescence microscopy. The replication kinetics and pathogenicity of the recombinant viruses are indistinguishable from the parental wild-type virus. This reverse genetics system will provide a powerful tool for the analysis of goose-origin NDV dissemination and pathogenesis, as well as preparation for genotype-matched NDV attenuated vaccines.

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.

Studies on construction of a recombinant Eimeria tenella SO7 gene expressing Escherichia coli and its protective efficacy against homologous infection.

Eimeria spp. are the causative agents of coccidiosis, a major disease affecting the poultry industry. A recombinant non-antibiotic Escherichia coli that expresses the Eimeria tenella SO7 gene was constructed and its protective efficacy against homologous infection in chickens was determined. The three-day-old chickens were orally immunized with the recombinant non-antibiotic SO7 gene expressing E. coli and boosted two weeks later. Four weeks after the second immunization, the chickens were challenged with 5 × 10(4) homologous sporulated oocysts. The protective effects of the recombinant non-antibiotic E. coli were determined by measuring body weight change, mortality, histopathology, lesion scores, oocyst counts, the specific antibody response and the frequency of CD4(+) and CD8(+) lymphocytes in peripheral blood. The results showed that immunization with SO7 expressing E. coli resulted in significantly improved body weight gain, reduced lesion scores and oocyst shedding in immunized chickens compared to controls. Furthermore, administration of recombinant SO7 expressing E. coli leads to a significant increase in serum antibody, CD4(+) and CD8(+) T cells in peripheral blood of chickens. These results, therefore, suggest that the recombinant non-antibiotic E. coli that expresses the SO7 gene is able to effectively stimulate host protective immunity as evidenced by the induction of development of both humoral and cell-mediated immune responses against homologous challenge in chickens.

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.

Generation and evaluation of a recombinant genotype VII Newcastle disease virus expressing VP3 protein of Goose parvovirus as a bivalent vaccine in goslings

Abstract Newcastle disease virus (NDV) and Goose parvovirus (GPV) are considered to be two of the most important and widespread viruses infecting geese. In this study, we generated a recombinant rmNA-VP3, expressing GPV VP3 using a modified goose-origin NDV NA-1 by changing the multi-basic cleavage site motif RRQKR↓F of the F protein to the dibasic motif GRQGR↓L as that of the avirulent strain LaSota as a vaccine vector. Expression of the VP3 protein in rmNA-VP3 infected cells was detected by immunofluorescence and Western blot assay. The genetic stability was examined by serially passaging 10 times in 10-day-old embryonated SPF chicken eggs. Goslings were inoculated with rmNA-VP3 showed no apparent signs of disease and developed a strong GPV and NDV neutralizing antibodies response. This is the first study demonstrating that recombinant NDV has the potential to serve as bivalent live vaccine against Goose parvovirus and Newcastle disease virus infection in birds.

Development and application of a blocking enzyme-linked immunosorbent assay (ELISA) to differentiate antibodies against live and inactivated porcine reproductive and respiratory syndrome virus.

The aim of this study was to establish a method that could differentiate antibodies against live and inactivated vaccines of porcine reproductive and respiratory syndrome virus (PRRSV). A blocking ELISA (b-ELISA) was established using the PRRSV non-structural protein, Nsp9, as the antigen and a monoclonal antibody, 2D6, against the Nsp9 protein as the capture antibody. The test was validated by using 415 clinical sera in the b-ELISA compared to a commercial kit based on the indirect ELISA using the nucleocapsid (N) protein as antigen. Significant differences were observed for the data obtained by the two detection methods. This may be due to the commercial kit detecting antibodies elicited by live and inactivated virus, whereas the b-ELISA only detects antibodies produced by any active viral replication, such as natural infection or live vaccination. Therefore, the b-ELISA in this study is able to distinguish between antibodies against live and inactivated viruses in pigs.