Improved immunogenicity of Newcastle disease virus inactivated vaccine following DNA vaccination using Newcastle disease virus hemagglutinin-neuraminidase and fusion protein genes

The present study describes the development of DNA vaccines using the hemagglutinin-neuraminidase (HN) and fusion (F) genes from AF2240 Newcastle disease virus strain, namely pIRES/HN, pIRES/F and pIRES-F/HN. Transient expression analysis of the constructs in Vero cells revealed the successful expression of gene inserts in vitro. Moreover, in vivo experiments showed that single vaccination with the constructed plasmid DNA (pDNA) followed by a boost with inactivated vaccine induced a significant difference in enzyme-linked immunosorbent assay antibody levels (p < 0.05) elicited by either pIRES/F, pIRES/F+ pIRES/HN or pIRES-F/HN at one week after the booster in specific pathogen free chickens when compared with the inactivated vaccine alone. Taken together, these results indicated that recombinant pDNA could be used to increase the efficacy of the inactivated vaccine immunization procedure.

Sendai F/HN Viroplexes for Efficient Transfection of Leukemic T Cells

PURPOSE: Most chemical transfection reagents are ineffective for the transfection of cells in suspension, such as leukemic cell and stem cell lineages. We developed two different types of viroplexes, cationic Sendai F/HN viroplexes (CSVs) and protamine sulfate-condensed cationic Sendai F/HN viroplexes (PCSVs) for the efficient transfection of T-leukemic cells. MATERIALS AND METHODS: The viroplex systems were prepared by reconstitution of fusogenic Sendai F/HN proteins in DMKE (O,O'-dimyristyl-N-lysyl glutamate) cationic liposomes. The viroplexes were further optimized for plasmid DNA and siRNA delivery to suspension cells. The particle size and surface charge of the viroplexes were analyzed with a zeta-sizer. Transfection of plasmid DNA (pDNA) and small interfering RNA (siRNA) by CSVs or PCSV was evaluated by measurement of transgene expression, confocal microscopy, FACS, and RT-PCR. RESULTS: The optimized CSVs and PCSVs exhibited enhanced gene and siRNA delivery in the tested suspension cell lines (Jurkat cells and CEM cells), compared with conventional cationic liposomes. In the case of pDNA transfection, the CSVs and PCSVs show at least 10-fold and 100-fold higher transgene expression compared with DMKE lipoplexes (or lipofectamine 2000), respectively. The CSVs showed more effective siRNA delivery to the suspension cells than cationic liposomes, as assessed by confocal microscopy, FACS, and RT-PCR. The effective transfection by the CSVs and PCSVs is presumably due to fusogenic activity of F/HN proteins resulting in facilitated internalization of pDNA and siRNA. CONCLUSION: This study suggests that Sendai F/HN viroplexes can be widely applicable for the transfection of pDNA and siRNA to suspension cell lines.

Structure-function analysis of two variants of mumps virus hemagglutinin-neuraminidase protein

A point mutation from guanine (G) to adenine (A) at nucleotide position 1081 in the hemagglutinin-neuraminidase (HN) gene has been associated with neurovirulence of Urabe AM9 mumps virus vaccine. This mutation corresponds to a glutamic acid (E) to lysine (K) change at position 335 in the HN glycoprotein. We have experimentally demonstrated that two variants of Urabe AM9 strain (HN-A1081 and HN-G1081) differ in neurotropism, sialic acidbinding affinity and neuraminidase activity. In the present study, we performed a structure-function analysis of that amino acid substitution; the structures of HN protein of both Urabe AM9 strain variants were predicted. Based on our analysis, the E/K mutation changes the protein surface properties and to a lesser extent their conformations, which in turn reflects in activity changes. Our modeling results suggest that this E/K interchange does not affect the structure of the sialic acid binding motif; however, the electrostatic surface differs drastically due to an exposed short alpha helix. Consequently, this mutation may affect the accessibility of HN to substrates and membrane receptors of the host cells. Our findings appear to explain the observed differences in neurotropism of these vaccine strains.

Protection of chickens from Newcastle disease with a recombinant baculovirus subunit vaccine expressing the fusion and hemagglutininneuraminidase proteins

Recombinant baculoviruses containing the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein gene of the viscerotropic velogenic (vv) Newcastle disease virus (NDV) isolate, Kr-005/00, and a lentogenic La Sota strain of the NDV were constructed in an attempt to develop an effective subunit vaccine to the recent epizootic vvNDV. The level of protection was determined by evaluating the clinical signs, mortality, and virus shedding from the oropharynx and cloaca of chickens after a challenge with vvNDV Kr-005/00. The recombinant ND F (rND F) and recombinant HN (rND HN) glycoproteins derived from the velogenic strain provided good protection against the clinical signs and mortality, showing a 0.00 PI value and 100% protection after a booster immunization. On the other hand, the combined rND F + HN glycoprotein derived from the velogenic strain induced complete protection (0.00 PI value and 100% protection) and significantly reduced the amount of virus shedding even after a single immunization. The rND F and rND HN glycoproteins derived from the velogenic strain had a slightly, but not significantly, greater protective effect than the lentogenic strain. These results suggest that the combined rND F + HN glycoprotein derived from vvNDV can be an ideal subunit marker vaccine candidate in chickens in a future ND eradication program.