Construction Of Bacterial Ghost Loading DNA Vaccine Of NDV F Gene And Its Evaluation As Vaccine Candidate

Newcastle disease (ND), caused by Newcastle disease virus (NDV), is a kind of highly contagious disease with worldwide distribution. NDV can infect many kinds of poultry such as chicken, turkeys, ducks, pigeons, parrots and cause much economic losses to the poultry industry, and the World Organization for Animal Health (namely OIE) determined ND as class I of animal infectious disease. Vaccination is still one of the main measures to control ND . DNA vaccine as a novel vaccine has many advantages, including non-pathogenity, easy to manufacture, non-interference from maternal antibody and becomes the focus of .the vaccine field. However, mass implementation in the field is hampered by the limit of administer routes, less immunogenic and high doses and/or multiple immunizations. Therefore much effort is now turned to increase the efficiency of DNA vaccine through: (i) using of adjuvants to enhance the immune response elicited by DNA vaccines, (ii) development of new delivery systems for specific targeting and/or better DNA uptake. The bacterial ghost system is a novel vaccine delivery system endowed with intrinsic adjuvant properties. Bacterial ghosts are nonliving Gram-negative bacterial cell envelopes devoid of cytoplasmic contents while maintaining their cellular morphology and native surface antigenic structures. The fact that bacterial ghosts preserve a native cell wall, including bioadhesive structures like fimbriae of their living counterparts, makes them suitable for the attachment to specific target tissues, moreover, its periplasmic and cytoplasmic space can accept many foreign matters, so it can be used as a effective tropic carrier and targeting tool. Based on all above mentioned, the main purpose of this study was to examine the immunogenicity of the DNA vaccine against NDV using bacterial ghost as a carrier for delivery of NDV F gene. The main contents are as follows:1. Construction of the eukaryotic plasmid expressing F gene of NDVAccording to the F gene sequence of NDV Lasota strain registered in GenBank (AF077761), the upstream and downstream primers were designed containing terminal BamH I and Kpn I restriction sites. The F gene was generated by RT-PCR amplification using the viral genome RNA as template. The PCR fragment about 1700bp was obtained, cloned into the vector pMD18-T to construct plasmid pT-F, introduced into DH5a competent cells by calcium shock method and plated on Luria-Bertani (LB) supplement with Ampicillin (50mg/ml), then the plasmid DNA was prepared for PCR and restriction enzyme digestion. The results of electrophoretic analysis suggested that the F gene was successfully cloned into vector. Subsequently, the fragment of F gene was inserted into the eukaryotic vector pcDNA3.1+ and identified with PCR and restriction enzyme digestion.2. Generation of DH5a ghosts by expression of lysis gene EBacterial ghosts are produced by expression of cloned gene E from bacteriophage PMX174 resulting cell lysis in Gram-negative bacteria, the lysis plasmid carries gene E under transcriptional control of the thermosensitiveλpL/pR-cI857 system. At 28℃or lower, gene E expression is stably repressed by repressor cI857, while was induced at temperatures higher than 30℃for inactivation of cI857, with optimal induction at 42℃Thus, for the production of bacterial ghosts, E. coli DH5a harboring the lysis plasmid pHH43 was grown at 28°C with agitation (200rpm). When the growing culture reached an optical density at 600 nm (OD600) of 0.4-0.6, the incubation temperature was shifted up to 42°C to induce the gene E-mediated lysis process. After another 4 h of incubation at 42°C with monitoring of the OD600, the culture was harvested. The efficiency of E-mediated killing of E. coli was estimated by plating samples of appropriate dilutions of freshly harvested ghosts on LB agar supplement with chloramphenicol, and the plates were incubated under culture conditions. Colonies were counted to determine the number of CFU, and the result indicated a 99.998% killing efficiency. Electron microscopic studies showed that the cytoplasmic content of the bacteria was expelled leaving an empty internal space and that the protein E-specific transmembrane tunnel structure, which permeabilized the bacterium was not randomly distributed over the cell envelope but was restricted to areas of potential division sites, predominantly in the middle of the cell or at polar sites. Except for the lysis hole, the morphology of the bacteria, including all cell surface structures and appendices, was not affected by the lysis event.3. Preparation of bacterial ghost loading DNA vaccine pcDNA3-FBacterial ghosts were resuspended in PBS buffer saline (pH7.4) containing pcDNA3-F, and after CaCl₂ supplemention (final concentration 25 mM), they were incubated with agitation (150rpm). For optimization of DNA concentration, bacterial ghosts were incubated with DNA concentrations ranging from 5 to 14mg/ml in PBS (pH7.4, 25 mM CaCl₂) at 37°C for 30 min. For the optimization of the incubation temperature, bacterial ghosts were incubated with the DNA (10 mg/ml in PBS, pH7.4, 25 mM CaCl₂) at 4, 25, and 37°C for 30 min until pelleted. For incubation time, bacterial ghosts were incubated with the DNA (10mg/ml in PBS, pH 7.4, 25 mM CaCl₂) at 24°C for 2, 4, 10, 30, 60, 120, and 180 min until pelleted. Incubation temperature, DNA concentration, and incubation time were varied systematically to optimize the loading procedure. The results demonstrated that the DNA concentration used for the loading was positively correlated with the amount of pcDNA3-F recovered from the bacterial ghosts. The DNA loads of bacterial ghosts were not significantly altered by incubation at temperatures of 4, 25, or 37°C. A 2-min incubation time was sufficient for the loading of the bacterial ghosts; longer incubation times (up to 180 min) did not increase the pcDNA3-F loading, as the DNA load stayed constant4. The evaluation of bacterial ghost loading the eukaryotic plasmid expressing NDV F geneOne hundred and eighty chickens with similar health condition were regarded as the experimental animals, which were randomly divided into 7 groups, with 30 chickens in each one at 7 day-old. I -II groups as the control were injected with PBS and pcDNA3.1 respectively, III group were injected with the naked pcDNA3-F, IV group were immunized with attenuated Lasota vaccine by eye-dropping, V-VII groups were administered with the bacterial ghost loading pcDNA3-F by eye-dropping, oral application or muscle injection respectively. Blood samples were collected weekly for indirect ELISA and HI assay before challenge. 2^nd week post booster immunization, chickens were challenged with the lethal dose of the isolated virulent NDV. 1^st week after primary immunization, the levels of antibody had risen slightly in pcDNA3-F group, attenuated Lasota vaccine group and bacterial ghost loading pcDNA3-F groups, compared with PBS group and pcDNA3.1+ group, which indicated that the DNA vaccine pcDNA3-F was successfully transformed and expressed into the chicken's cell, thereby stimulated efficient immune response. 2^nd week after primary immunization, compared with control groups, the levels of all groups except control groups had risen obviously (P<0.01). What's more, at 1^st week post booster immunization, the pcDNA3-F-loaded ghost by eye-dropping group was higher than the naked pcDNA3-F group (P<0.05), and significantly higher at 2^nd week post booster immunization (P<0.01), although it was lower than the attenuated Lasota vaccine group. In the same period, the antibody level of the attenuated Lasota vaccine group kept the highest in all vaccination groups, and the antibody level of the control groups descent continuously. The result of HI suggested that the antibody induced by DNA vaccine pcDNA3-F had not relation with the antibody of HI. The protective immunization score was 70% for the group III chickens that were inoculated with the bacterial ghost loading pcDNA3-F vaccine, which was higher than the naked pcDNA3-F group, while the group that received the attenuated Lasota vaccine had 90% protection. 100% fatality was recorded for the control groups (PBS and pcDNA3.1+). The remaining two groups receiving bacterial ghost loading pcDNA3-F either by orally or i.m, whose mortality was same as the naked pcDNA3-F group, but delayed the time of mortility. These results suggested that the DNA vaccine pcDNA3-F could induce more effective immune response against NDV, when used bacterial ghosts as a delivery system...