Search

Epitope-based vaccination might play an important role in the protective immunity against Japanese encephalitis virus (JEV) infection. The purpose of the study is to evaluate the immune characteristics of recombinant MVA carrying multi-epitope gene of JEV (rMVA-mep). The synthetic gene containing critical epitopes (B-cell, CTL and Th) of JEV was cloned into the eukaryotic expression vector pGEM-K1L, and the rMVA-mep was prepared. BALB/c mice were immunized with different dosages of purified rMVA-mep and the immune responses were determined in the form of protective response against JEV, antibodies titers (IgG1 and IgG2a), spleen cell lymphocyte proliferation, and the levels of interferon-γ and interleukin-4 cytokines. The results showed that live rMVA-mep elicited strongly immune responses in dose-dependent manner, and the highest level of immune responses was observed from the groups immunized with 107 TCID50 rMVA-mep among the experimental three concentrations. There were almost no difference of cytokines and neutralizing antibody titers among 107 TCID50 rMVA-mep, recombinant ED3 and inactivated JEV vaccine. It was noteworthy that rMVA-mep vaccination potentiates the Th1 and Th2-type immune responses in dose-dependent manner, and was sufficient to protect the mice survival against lethal JEV challenge. These findings demonstrated that rMVA-mep can produce adequate humoral and cellular immune responses, and protection in mice, which suggested that rMVA-mep might be an attractive candidate vaccine for preventing JEV infection.

This article proposes a novel equilibrium finite element method (EFEM) for equilibrated solution of frictional contact problem. The development of such EFEM is mainly two‐fold. On the one hand, the traction‐based equilibrium element is employed to construct the discrete equilibrated stress field. This equilibrium element, using edge tractions as basic variables, is found straightforward to deal with the friction and contact constraints on the contact surface. On the other hand, the Coulomb frictional contact problem is recast into a linear complementarity problem (LCP), with which the algebraic complementarity formulation can be obtained with the help of the constructed equilibrated stress field. A remarkable property of the proposed EFEM is that the equilibrated solution, in conjunction with the compatible solution of the traditional FEM, can provide reliable error estimation for frictional contact within the framework of dual analysis. Numerical examples are finally conducted to see the performance and the ability to reliable error estimation of the proposed EFEM. [ABSTRACT FROM AUTHOR]

Abstract: Hydrogen sulfide (H2S), an endogenous gaseous mediator, has been shown to exert protective effects against damage to different organs in the human body caused by various stimuli. However, the potential effects of H2S on hypoxia-induced neuronal apoptosis and its mechanisms remain unclear. Here, we exposed mouse hippocampal neurons to hypoxic conditions (2% O2, 5% CO2 and 93% N2 at 37°C) to establish a hypoxic cell model. We found that 4-h hypoxia treatment significantly increased intracellular reactive oxygen species (ROS) levels, and pretreatment with NaHS (a source of H2S) for 30min suppressed hypoxia-induced intracellular ROS elevation. The hypoxia treatment significantly increased cytosolic calcium ([Ca2+]i), and pretreatment with NaHS prevented the increase in [Ca2+]i. Additionally, polyethylene glycol (PEG)–catalase (a H2O2 scavenger) but not PEG–SOD (an O2 - scavenger) conferred an inhibitory effect similar to H2S on the hypoxia-induced increase in [Ca2+]i. Furthermore, we found that pretreatment with NaHS could significantly inhibit hypoxia-induced neuronal apoptosis, which was also inhibited by PEG-catalase or the inositol 1,4,5-triphosphate (IP3) receptor blocker xestospongin C. Taken together, these findings suggest that H2S inhibits hypoxia-induced apoptosis through inhibition of a ROS (mainly H2O2)-activated Ca2+ signaling pathway in mouse hippocampal neurons. [Copyright &y& Elsevier]

Based on gram positive enhancer matrix displaying technology, we designed and evaluated a bacteria-like particle vaccine against swine type O Foot-and-mouth disease virus. Three optimized genes of type O Foot-and-mouth disease virus strain Mya98 were cloned into recombinant prokaryotic expression vector pQZ-PA and renamed as pQZ-BT1B-PA, pQZ-BT2B-PA and pQZ-B (T1BT2) 4B-PA, fused with an anchor protein (PA) binding to Gram-positive enhancer matrix (GEM) particles specifically. The protein expression was identified with SDS-PAGE and Western blotting, and then purified with GEM particles. Five-week old female mice were randomly divided into six groups and all the immunization was developed according to subcutaneous injection. Mice in the first three groups were injected with 50 μg/dose GEM-BT1B, GEM-BT2B and GEM-B (T1BT2) 4B, respectively. Mice in the fourth group were immunized with commercial peptide vaccine as positive control. The fifth group vaccinated with host E. coli transformed with pQZ-PA fulfilled as negative control. Mice in the last group injected with sterile PBS served as blank control. The humoral immunity of recombinant protein vaccine was evaluated with peptide-specific antibody and LPB antibody. The cellular immunity was evaluated with lymphocyte proliferation test and cytokine expression detection. SDS-PAGE and Western blotting showed that the most part of soluble target fusion protein have been purified and displayed on GEM particles. Vaccine GEM-B (T1BT2) 4B stimulated mice produce not only higher level of specific antibody against peptide and Foot-and-mouth disease virus specific liquid phase blocking antibody, but also more vigorous spleen lymph proliferation and higher levels of Th1 type cytokines. To summarize, vaccine of GEM-B (T1BT2) 4B possessed good immunogenicity and opened a new way for further Foot-and-mouth disease virus subunit vaccine design.

Objective: The aim of the present study was to compare the binding activity of Lactococcus lactis peptidoglycan protein anchor (PA) with different number of motifs., Methods: L. lactis PA gene sequences with 2 and 3 lysin motifs (LysM) were obtained by PCR amplification. Then, the recombinant plasmid of pET-32a(+) containing PA gene was constructed and transformed into E. coli BL21 (DE3) , and induced to express the fusion protein PA2 and PA3. The purified and refolded PA2 and PA3 were incubated with gram positive enhancer matrix (GEM). The binding activities of PA2 and PA3 were identified and compared by Western blot, TEM and SDS-PAGE., Results: The PA2 and PA3 could bind to GEM. The anchoring activity of PA3 was obviously superior to the PA2., Conclusion: The data indicated that PA with 3 LysMs had a better binding capacity compared with 2 LysMs. The results provided foundations to further improve the design of GEM-PA display system.

Unlabelled: A novel double expression shuttle vector named pLR-gpt was constructed for marker-free recombinant modified vaccinia virus Ankara generation. A delectable Eco gpt marker was adopted with Cre/LoxP DNA recombination system and a BHK-21 cell line that can express Cre enzyme. Eco gpt gene controlled by P7.5 promoter from Vaccinia virus was cloned between two LoxP sites in the same direction. Additionally, two multiple cloning site under control of other two Vaccinia virus promoters were constructed outside LoxP sites. With this new transfer vector, Eco gpt marker in rMVA can be deleted on BHK-Cre with interaction between Cre enzyme and LoxP sequence. In order to verify the efficacy of this system, ORF5 and ORF6 gene of Porcine reproductive and respiratory syndrome virus (PRRSV) NJ-a strain were cloned into two multiple cloning sites of pLR-gpt to construct recombinant plasmid pLR-ORFS/ORF6. Homologous recombination between pLR-ORF5/ORF6 and wtMVA on BHK-21 cell was mediated by liposome by infecting cells with 0.01 MOI wtMVA two hours before transfection. After twelve cycles of selection, recombinant MVA with selecting marker Eco gpt was obtained and named as rMVAgpt-GP5/M. By infecting BHK-Cre, the Eco gpt marker in rMVAgpt-GP5/M was deleted and this rMVA was named as rMVA-GP5/M. Expression of GP5 and M protein was identified with Western blotting and IFA. Results from PCR and biological study for rMVA indicated that Eco gpt marker was completely deleted., Conclusions: double expression transfer vector for marker-free recombinant Modified vaccinia virus Ankara generation was successfully constructed, and works well in MVA expression system.

In order to obtain a virus-like particle vaccine both for porcine parvovirus (PPV) prevention and growth-promotion, VP2 gene of PPV NJ-a strain was amplified with PCR, and four copies of synthetic somatostatin gene were fused to the N-terminal of VP2 gene. The fused gene was cloned into pFast-HT A to construct the recombinant plasmid pFast-SS4-VP2, then the pFast-SS4-VP2 was transformed into DH10Bac competent cells and recombined with shuttle vector Bacmid, followed by identification with blue-white screening and PCR analysis for three cycles, and the positive recombinant was named as rBacmid-SS4-VP2. The positive Sf-9 cells were transfected with rBacmid-SS4-VP2 by Lipofectamine to produce recombinant baculovirus. When the cytopathic effect (CPE) was obvious, the transfected Sf-9 cell was harvested, and the positive recombinant virus was named as rBac-SS4-VP2. The insertion for the target gene into baculovirus genome was confirmed with PCR. SDS-PAGE and Western blotting revealed that the calculated protein of approximately 68 kDa was in the expressed in the insect cells. The Sf-9 cells infected with rBac-SS4-VP2 were stained positive against PPV antibody using the indirect immunofluorescence assay (IFA). Moreover, the virus particle self-assembly was observed under electron microscopy. 90 four-week-old mice were immunized by the recombinant protein coupled with different adjuvants alhydrogel, IMS and oil. VP2-specific ELISA antibodies, PPV-specific neutralizing antibody, somatostatin antibody and growth hormone levels were examined to evaluate the immunogenicity of this virus like particle. Results indicated that mice groups immunized rSS4-VP2 protein with alhydrogel and IMS developed similar humoral immune response comparing with inactived PPV vaccine. Mice group immunized with rSS4-VP2 generated higher level of SS antibody and growth hormone comparing with negative control, mice receiving rSS4-VP2 with alhydrogel developed the highest antibody titre than all other groups, while the oil group developed the lowest antibody level. This study provides not only a new rout for production of safe and effective virus like particle subunit vaccine, but also the foundations for peptide presentation and multivalent subunit vaccine design.

Objective: In order to overcome the defect of traditional avian influenza vaccine that lacks cross-protection among different serotypes, we developed a universal anti-influenza vaccine., Methods: Based on the gene analysis for A IV matrix protein 2 and two cytotoxic T-lymphocyte epitopes, we constructed four prokaryotic expression vectors. The target gene was induced by IPTG and the fusion protein was mixed with Freund's adjuvant; then used to immunize 20-day-old chicken by intramuscular injection and boosted 3 weeks later. Blood samples were collected weekly following the primary vaccination. The anti-M2e antibody was detected with ELISA coated by synthesized peptide; the neutralizing ability of anti-serum was evaluated on MDCK cell line and chick embryo; the CD4+ and CD8+ T lymphocyte amounts in peripheral blood of immunized chicken were measured by flow cytometry., Results: The fusion protein induced immunological reaction, and the antibody bound with the viral M2 protein expressed on the surface of MDCK cells. Serum could only inhibit the replication without neutralizing the virus. Flow cytometry results showed that CD4+ and CD8+ T lymphocyte in peripheral blood increased obviously following immunization (P<0.05), which possessed the character of cell immunization., Conclusion: Chimeric peptides kept good immuno-genicity and provided useful probes for the control of avian influenza.

Modified ORF5 (MORF5) and ORF6 gene of PRRSV were cloned into two multiple cloning sites of MVA transfer vector pLR-gpt to construct the recombinant plasmid pLR-MORF5/ORF6. Homologous recombination between pLR-MORF5/ORF6 and the wtMVA on BHK-21 cell line was mediated with liposome by infecting the cell with 0.01 MOI wtMVA two hours before transfecting the recombinant plasmid into the cell. When the cytopathic effect (CPE) was obvious, virus was collected from the cell plate and the recombinant virus was selected with drug selecting medium (2% MXHAT). After 12 cycles of selection, rMVA with a selection marker Eco gpt was obtained and named as rMVAgpt-MGP5/M. By infecting BHK-Cre expressing Cre recombinant enzyme, the Eco gpt marker in rMVAgpt-MGP5/M was deleted and this rMVA was named as rMVA-MGP5/M. The insertion of MORF5 and ORF6 into the MVA genome was confirmed with PCR analysis and the expression of MGP5 and M protein was identified with Western blot and IFA. Through biological study on the recombinant MVA, no obvious difference was observed between rMVA-MGP5/M and the wtMVA regarding to the CPE and growth curve. The recombinant MVA constructed in this study could coexpress the modified GP5 and M protein and the expressed product had good immunocompetence. Furthermore, the insertion of the MORF5 and ORF6 into MVA genome had no obvious effect on the replication and biological characteristics of this virus.