Attenuated Salmonella Typhimurium As A Transgenic Carrier For Prokaryotic And Eukaryotic Expression, And Newcastle Disease Virus Surface Glycoproteins Naked DNA Immunization

Progress in biotechnology and molecular biology has produced a paradigm shift in vaccinology. Such a shift has occurred with the advent of DNA-mediated immunization, now colloquially known as DNA vaccines. This new concept of immunization entails the direct introduction of essentially a plasmid DNA harboring target genes into various tissues of an organism using needle injection or particle bombardment. Naked DNA vaccines have been found effective against a variety of pathogens by elicitation of both arms of the immune system, the humoral and cellular immune responses. Independent trials demonstrated that DNA vaccines targeting the fusion protein gene (F) of Newcastle disease virus (NDV) induced immune responses or provide protection of chickens with against lethal challenge with virulent strains. However, immunization of chickens with recombinant plasmid of NDV HN gene encoding hemagglutinin-neuraminidase or co-administration of both recombinant plasmids of NDV-F plus NDV-HN is lacking or limited in the annals of "third generation vaccine" records.Newcastle disease is one of the most serious infectious diseases of poultry. It is a highly contagious disease with worldwide distribution, causing significant economic losses in the commercial poultry industry and village chickens in developing countries. NDV, the causative agent of Newcastle disease, is the prototype virus for Type 1 avian paramyxoviruses. The virus is an enveloped virus with a negative sense single-stranded RNA genome of 15,186 kb which codes for an RNA directed RNA polymerase, surface glyccoproteins (hemagglutinin-neuraminidase protein and fusion protein), matrix protein, phosphoprotein and nucleoprotein in the 5' to 3' direction The HN protein is important in attachment and release of the virus from the host cells, while F protein has a critical role in pathogenesis by mediating viral fusion to host cell membrane.In the present study, the HN gene of NDV strain F48E9 was amplified from the viral genome RNA by RT-PCR and cloned directionally into eukaryotic vector pcDNA₃ under the control of human cytomegalovirus (hCMV) immediate early enhancer and. The HN gene was sequenced from the putative recombinant PCDNA3-HN as having 1716 nucleotides. A single open reading frame in the sequence encodes a protein of 570 amino acids with a calculated molecular weight of 75,200.Comparison of the F48E9 HN protein sequence with 10 other paramyxoviruses previously published sequences showed more than 92% homology.The ability of the constructed vaccines to express full-length antigens in vitro was investigated in HeLa Cells. Bactofections of cells with attenuated S. typhimurim and cationic liposome mediated transfection of HeLa cells were employed in eukaryotic expression of the reporter genes used in this studies. SDS-PAGE and Western blot revealed a band of 75 kDa from cell lysates suggesting a immunoreactive HN protein is expressed. The band is consistent with the predicted size in published reports. Furthermore, expression of eGFP in HeLa cells using attenuated S. typhimurim as a transgenic carrier also validated the competence of the system. The F gene used in this experiment was earlier cloned and expressed in eukaryotic cells. These findings suggest that the DNA vaccines could be used for trials in chickens against virulent NDV strains challenge.In a separate experimental setting, attenuated Salmonella enterica serovar typhimurium strain as the host for recombinant prokaryotic and eukaryotic vectors containing the enhanced green fluorescent proteins (eGFP) gene was examined by fluorimetric and fluorescent microscopic methods. Expression of eGFP was dependent on the concentration of IPTG used for induction, being optimal at 0.5 mM. Further increase to 0.75 mM did not increase the fluorescence output. This dose-dependent induction was not apparent when E. coli was used as the host strain. The expression was higher in S. typhimurium than in E. coli especially when the inducer concentration was at or above 0.5mM. The attenuated S. typhimurium strain was invasive, though less than its parent strain, into the HeLa cells and able to deliver the recombinant eukaryotic plasmid pcDNA3-eGFP for expression of eGFP as shown by fluorescing cells 48 hrs after transfection. The results of this experiment also demonstrate the utility of direct measurement of fluorescence and optical density in a multifunctional microplate-based spectrophotpmetric reader, allowing high throughput multiple quantitative comparisons of eGFP expression by different host strains or the same strain under different conditions or even different expression vectors.The efficacy of the naked NDV vaccines pcDNA3-HN, pcDNA3-F and pcDNA3-HN + pcDNA3-F was evaluated in 1-week old specific pathogen free chicks divided into groups I, II and III receiving corresponding recombinant plasmids. Four control groups were also included the experiment (pcDNA3, PBS challenge group,PBS non-challenge group and a regular commercial attenuated NDV vaccine strain vaccinated group for comparison). 150 ug of DNA in lOOul PBS was administered per chick in groups I and II, while group HI received 300 ug/chick (combined doses of groups I and II). Two weeks later all groups were boosted at the same dose levels. Blood samples were collected at different points before challenge for antibody titration and for lymphocyte proliferation assay (LPA). Chickens were challenged with a lethal dose of NDV F4gE9 strain 4 weeks after the booster immunization.With P/N value > 2, only chicks immunized with NDV commercial stains vaccine at second week after first vaccination had a rise in titer that was significant and in the second week after booster dose. Group vaccinated with the combination of recombinant plasmid (pcDNA3-HN + pcDNA3-F) had significant rise in antibody titer. The same trend was observed at fourth week after booster dose. Surprisingly, groups vaccinated with pcDNA3-F or pcDNA3-HN only had rise in antibody titer in at week 4 after boosting immunization. A significant increase (P< 0.05) in lymphocyte proliferation could be detected for all the groups at four weeks post second vaccination except for PBS challenge and pcDNA3 groups. The protective immunization score was 41% for the group HI chickens that were inoculated with the combined vaccine (pcDNA3-HN + pcDNA3-F), while the group that received the attenuated NDV commercial vaccine had 82% protection. 100% mortality was recorded for the PBS control group and the remaining two groups receiving either pcDNA3-F or pcDNA3-HN alone, though there were increased antibody titers for these two groups at four weeks post booster dose. These results suggest that naked DNA vaccines pcDNA3-F and pcDNA3-HN were capable of eliciting immune responses but offered no protection when used alone. However, the co-administration of both plasmids synergjstically boosted the immune responses to a level of protection of chickens against virulent NDV challenge.