Towards the Development of DNA Vaccine Against Newcastle Disease Virus

The etiological agent of Newcastle disease, Newcastle disease virus (NDV), a member of the family Paramyxoviridae and the genus of Rubulavirus, can cause up to 100% morbidity and mortality. Immune responses to both the fusion (F) and haemagglutinin-neuraminidase (HN) protein antigens of NDV were...

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Main Author:	Loke, Chui Fung
Format:	Thesis
Language:	English English
Published:	2002
Subjects:	Newcastle disease virus DNA vaccines
Online Access:	http://psasir.upm.edu.my/id/eprint/8480/1/FSMB_2002_17_IR.pdf
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spelling	my-upm-ir.84802024-01-26T07:45:20Z Towards the Development of DNA Vaccine Against Newcastle Disease Virus 2002-08 Loke, Chui Fung The etiological agent of Newcastle disease, Newcastle disease virus (NDV), a member of the family Paramyxoviridae and the genus of Rubulavirus, can cause up to 100% morbidity and mortality. Immune responses to both the fusion (F) and haemagglutinin-neuraminidase (HN) protein antigens of NDV were demonstrated to play an important role in the prevention of infection. Towards development of DNA vaccine, both the F and HN genes of a Malaysian heat resistant viscerotropicvelogenic NDV strain AF2240 were amplified and cloned into a mammalian expression vector, pEGFP-Ns, and expressed in a mammalian cell line under the control of the immediate early promoter of human cytomegalovirus. Six recombinant plasmids were constructed, namely pEGFP-N3/F, -N1/HN, - N3/HN-GFP, -N2/Fkoz, -N2/HNkoz and -N1/Fkoz-GFP with the later three constructs introduced with the kozak translation initiation sequences. Transient expression of F and HN proteins was assayed in vitro in Vero cell at 48 h posttransfection by indirect immunofluorescence using NDV polyclonal antibody and fluorescein isothiocyanate (FITC)-labelled anti-chicken IgG. The results showed that all the DNA-transfected cells exhibited bright cytoplasmic fluorescene, indicating both the F and HN proteins were successfully expressed in the mammalian cell line. Immunoblot analysis of the transfected cell lysates further verified the presence of the recombinant proteins with a distinct band of 64 kDa which corresponds to the uncleaved precusor Fo glycoprotein of NDV and two bands of -62 and 72 kDa as unglycosylated and glycosylated HN glycoproteins, respectively. eSS]-methionine pulsed labelling of transfected cells confirmed the expression of green fluorescent protein (GFP)-fusion protein of F, but not HN-GFP. DNA inoculation in Balb/c mice and specific pathogen free (SPF) chicken revealed that the efficacy of DNA vaccines could be boosted by co-administration of Freund's adjuvant and repeating DNA immunization. The vaccine trial in SPF chickens showed that both the circular and linearized plasmid DNA of pEGFP-N3IF produced significant levels of antibody against NDV after the second booster and conferred 40-47% protection upon lethal NOV challenge. Co-administration of the circular plasmids of pEGFP-N3/F and -NIIHN, produced antibodies efficiently and conferred more than 50% protection upon NOV challenge. The low and undetectable antibody level in some of the survivors suggests that DNA vaccine elicits cellular immune response in chicken. The overall results also suggest that both the F and HNDNA can be used as a vaccine component to provide effective protection against NDV and DNA immunization opens a new approach to the development of gene vaccine for chicken against infectious disease. Newcastle disease virus DNA vaccines 2002-08 Thesis http://psasir.upm.edu.my/id/eprint/8480/ http://psasir.upm.edu.my/id/eprint/8480/1/FSMB_2002_17_IR.pdf text en public doctoral Universiti Putra Malaysia Newcastle disease virus DNA vaccines Food Science and Technology Abdul Rahim, Raha English
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English English
advisor	Abdul Rahim, Raha
topic	Newcastle disease virus DNA vaccines
spellingShingle	Newcastle disease virus DNA vaccines Loke, Chui Fung Towards the Development of DNA Vaccine Against Newcastle Disease Virus
description	The etiological agent of Newcastle disease, Newcastle disease virus (NDV), a member of the family Paramyxoviridae and the genus of Rubulavirus, can cause up to 100% morbidity and mortality. Immune responses to both the fusion (F) and haemagglutinin-neuraminidase (HN) protein antigens of NDV were demonstrated to play an important role in the prevention of infection. Towards development of DNA vaccine, both the F and HN genes of a Malaysian heat resistant viscerotropicvelogenic NDV strain AF2240 were amplified and cloned into a mammalian expression vector, pEGFP-Ns, and expressed in a mammalian cell line under the control of the immediate early promoter of human cytomegalovirus. Six recombinant plasmids were constructed, namely pEGFP-N3/F, -N1/HN, - N3/HN-GFP, -N2/Fkoz, -N2/HNkoz and -N1/Fkoz-GFP with the later three constructs introduced with the kozak translation initiation sequences. Transient expression of F and HN proteins was assayed in vitro in Vero cell at 48 h posttransfection by indirect immunofluorescence using NDV polyclonal antibody and fluorescein isothiocyanate (FITC)-labelled anti-chicken IgG. The results showed that all the DNA-transfected cells exhibited bright cytoplasmic fluorescene, indicating both the F and HN proteins were successfully expressed in the mammalian cell line. Immunoblot analysis of the transfected cell lysates further verified the presence of the recombinant proteins with a distinct band of 64 kDa which corresponds to the uncleaved precusor Fo glycoprotein of NDV and two bands of -62 and 72 kDa as unglycosylated and glycosylated HN glycoproteins, respectively. eSS]-methionine pulsed labelling of transfected cells confirmed the expression of green fluorescent protein (GFP)-fusion protein of F, but not HN-GFP. DNA inoculation in Balb/c mice and specific pathogen free (SPF) chicken revealed that the efficacy of DNA vaccines could be boosted by co-administration of Freund's adjuvant and repeating DNA immunization. The vaccine trial in SPF chickens showed that both the circular and linearized plasmid DNA of pEGFP-N3IF produced significant levels of antibody against NDV after the second booster and conferred 40-47% protection upon lethal NOV challenge. Co-administration of the circular plasmids of pEGFP-N3/F and -NIIHN, produced antibodies efficiently and conferred more than 50% protection upon NOV challenge. The low and undetectable antibody level in some of the survivors suggests that DNA vaccine elicits cellular immune response in chicken. The overall results also suggest that both the F and HNDNA can be used as a vaccine component to provide effective protection against NDV and DNA immunization opens a new approach to the development of gene vaccine for chicken against infectious disease.
format	Thesis
qualification_level	Doctorate
author	Loke, Chui Fung
author_facet	Loke, Chui Fung
author_sort	Loke, Chui Fung
title	Towards the Development of DNA Vaccine Against Newcastle Disease Virus
title_short	Towards the Development of DNA Vaccine Against Newcastle Disease Virus
title_full	Towards the Development of DNA Vaccine Against Newcastle Disease Virus
title_fullStr	Towards the Development of DNA Vaccine Against Newcastle Disease Virus
title_full_unstemmed	Towards the Development of DNA Vaccine Against Newcastle Disease Virus
title_sort	towards the development of dna vaccine against newcastle disease virus
granting_institution	Universiti Putra Malaysia
granting_department	Food Science and Technology
publishDate	2002
url	http://psasir.upm.edu.my/id/eprint/8480/1/FSMB_2002_17_IR.pdf
_version_	1794018757764448256

Towards the Development of DNA Vaccine Against Newcastle Disease Virus

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