| Recent studies show that attenuated intracellular bacteria can be used for delivery of DNA vaccines. Salmonella typhimuriwn,one of the invasive bacterial species,can be attenuated without loss of invasiveness and thus used for delivery of eukaryotic expression vectors into host cells in vivo. The recombinant plasmid containing the target gene is released inside the host cells and gain entry into the nucleus,resulting in expression of encoded antigens and subsequent induction of humoral and cellular immune responses. However,there has been no report on genetic vaccination against avian diseases using S. typhimurium as the oral delivery vector for DNA vaccines.Newcastle disease,a highly contagious disease with worldwide distribution,can cause severe economic losses to the poultry industry. The causative agent of the disease,Newcastle disease virus (NDV),is a member of the Paramyxoviridae whose genome is a non-segmented single-strand negative-sense RNA. The fusion (F) protein and hemagglutinin-neuraminidase (HN) are the major virulence factors of the virus to which the host responds well with protective antibody production. Recombinant plasmids containing the F or HN gene can induce specific immune responses against NDV. However,DNA vaccination by injection or gene gun routes is rather inconvenient and costly in field applications in scaled animal farms. Oral delivery of nude DNA failed to induce enough immune response in chickens. The main purpose of this study was to examine the immunogenicity of a DNA vaccine against NDV using the attenuated S. typhimurium with dam and phoP mutations as a vector for delivery of the NDV F gene.The full-lengh cDNA of the F gene of a virulent NDV strain F4gE9 was amplified by RT-PCR and inserted into pcDNAS under the control of human cytomegalovirus (hCMV) immediate early enhancer and promoter. The F gene from the resulting recombinant plasmid pcDNA3-F was sequenced. The gene was 1668bp in length,encoding the F protein composed of 553 amino acids. Sequence analysis and its secondary structure prediction showed that the F protein had three major hydrophobic regions consisting of about 25 amino acids,six potential glycosylation sites and thirteen cysteines. A sequence region of basic amino acids,RRQRRF,was found at the F,-F2 cleavage site,indicating that F4gE9 was a typical virulent strain. The strain F48E9 shared 91.3 %-95.5 % homology with other NDVstrains of chicken origin at the amino acid level,related most closely to the strain Australia-victoria with 95.5 % homology,but a little distant from the strains Ch/98-1a of pigeon origin and ZJla of goose origin with 90.6 % homology.The recombinant eukaryotic expression plasmid pcDNA3-F was transformed by electroporation into an attenuated S. typhimurium strain ZJ111(dam" and phoP"),resulting in ZJ111/ pcDNA3-F which was then used to infect the Vero cells. DNA and RNA dot blotting revealed that the F gene was transcribed into mRNA in the Vero cells. There was expression of the F protein as shown by indirect immunofluorescent assay. The expression began at 48h post-infection and increased thereafter,as indicated by ELISA. A 55 KD band of the F protein was identified by SDS-PAGE and Western blotting. These results clearly showed that the expressed F protein was immune-reactive with chicken anti-NDV serum.Tests for safety and stability indicated that ZJ111/ pcDNA3-F as the oral NDV DNA vaccine was of low pathogenicity to mice with only a few mice dead at a high dose of 109 cfu and no death with other groups dosed at or below 10s cfu. It did not cause any death or side effects on chickens orally dosed at or below 108 cfu. At week 2 after the 2nd immunization,no S. typhimurium cells were recovered from the spleen and liver. ZJ111/pcDNA3-F showed high stability in vitro and in vivo even in the absence of ampicillin as determined by enzymatic digestion and PCR.Three-day-old non-immunized chickens were immunized orally with ZJ111/pcDNA3-F at three dosage levels (109cfu,108cfu and 107cfu) and boosted two weeks later with th... |
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