Expression, Purification And Quantitation Of Hemagglutinin-Neuraminidase For Dose-Ranging Assessment Of A Recombinant Protein-Based Newcastle Disease Subunit Vaccine

Recombinant hemagglutinin-neuraminidase (HN) based subunit vaccine, which is non-infectious and can be produced using insect cell-culture systems, is a potential alternative to conventional live and inactivated Newcastle disease (ND) vaccines. However, process development for manufacture and efficac...

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Main Author:	Lye, Ping Ying
Format:	Thesis
Language:	English
Published:	2020
Subjects:	R5-920 Medicine (General)
Online Access:	http://eprints.usm.my/52168/1/LYE%20PING%20YING%20-%20TESIS%20cut.pdf
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spelling	my-usm-ep.521682022-04-04T08:32:19Z Expression, Purification And Quantitation Of Hemagglutinin-Neuraminidase For Dose-Ranging Assessment Of A Recombinant Protein-Based Newcastle Disease Subunit Vaccine 2020-05 Lye, Ping Ying R5-920 Medicine (General) Recombinant hemagglutinin-neuraminidase (HN) based subunit vaccine, which is non-infectious and can be produced using insect cell-culture systems, is a potential alternative to conventional live and inactivated Newcastle disease (ND) vaccines. However, process development for manufacture and efficacy assessment of HN based subunit vaccines has been hampered by the absence of reference standards, a cornerstone for robust and sensitive quantitative analytical methods. In this work, a downstream purification strategy was developed to obtain NDV HN which was expressed with a hexa-histidine fusion tag (rHN) to facilitate detection using generic antibodies. Highly purified rHN (~95%) attained after detergent extraction and two-stage ion-exchange-hydroxyapatite column chromatography was subsequently utilized as reference standards for quantitative ELISA development. The calibration curve for the developed ELISA was found to be linear between the range of 15.6‒1000 ng mL−1. The intra- and inter-assay precision expressed as a coefficient of variation (CV) were less than 10% and 12% respectively. Recovery of rHN at different stages of purification was monitored. Quantitation of rHN from crude cell lysates was subsequently performed for dose-ranging antibody response and protective efficacy studies. A higher dose (1500 ng) of rHN was correlated to a significant reduction in virus shedding and attainment of herd immunity, as indicated by a higher proportion of chickens (92%) with hemagglutination inhibition (HI) antibody titers ≥log23. 2020-05 Thesis http://eprints.usm.my/52168/ http://eprints.usm.my/52168/1/LYE%20PING%20YING%20-%20TESIS%20cut.pdf application/pdf en public masters Universiti Sains Malaysia Institut Penyelidikan Perubatan Molekul
institution	Universiti Sains Malaysia
collection	USM Institutional Repository
language	English
topic	R5-920 Medicine (General)
spellingShingle	R5-920 Medicine (General) Lye, Ping Ying Expression, Purification And Quantitation Of Hemagglutinin-Neuraminidase For Dose-Ranging Assessment Of A Recombinant Protein-Based Newcastle Disease Subunit Vaccine
description	Recombinant hemagglutinin-neuraminidase (HN) based subunit vaccine, which is non-infectious and can be produced using insect cell-culture systems, is a potential alternative to conventional live and inactivated Newcastle disease (ND) vaccines. However, process development for manufacture and efficacy assessment of HN based subunit vaccines has been hampered by the absence of reference standards, a cornerstone for robust and sensitive quantitative analytical methods. In this work, a downstream purification strategy was developed to obtain NDV HN which was expressed with a hexa-histidine fusion tag (rHN) to facilitate detection using generic antibodies. Highly purified rHN (~95%) attained after detergent extraction and two-stage ion-exchange-hydroxyapatite column chromatography was subsequently utilized as reference standards for quantitative ELISA development. The calibration curve for the developed ELISA was found to be linear between the range of 15.6‒1000 ng mL−1. The intra- and inter-assay precision expressed as a coefficient of variation (CV) were less than 10% and 12% respectively. Recovery of rHN at different stages of purification was monitored. Quantitation of rHN from crude cell lysates was subsequently performed for dose-ranging antibody response and protective efficacy studies. A higher dose (1500 ng) of rHN was correlated to a significant reduction in virus shedding and attainment of herd immunity, as indicated by a higher proportion of chickens (92%) with hemagglutination inhibition (HI) antibody titers ≥log23.
format	Thesis
qualification_level	Master's degree
author	Lye, Ping Ying
author_facet	Lye, Ping Ying
author_sort	Lye, Ping Ying
title	Expression, Purification And Quantitation Of Hemagglutinin-Neuraminidase For Dose-Ranging Assessment Of A Recombinant Protein-Based Newcastle Disease Subunit Vaccine
title_short	Expression, Purification And Quantitation Of Hemagglutinin-Neuraminidase For Dose-Ranging Assessment Of A Recombinant Protein-Based Newcastle Disease Subunit Vaccine
title_full	Expression, Purification And Quantitation Of Hemagglutinin-Neuraminidase For Dose-Ranging Assessment Of A Recombinant Protein-Based Newcastle Disease Subunit Vaccine
title_fullStr	Expression, Purification And Quantitation Of Hemagglutinin-Neuraminidase For Dose-Ranging Assessment Of A Recombinant Protein-Based Newcastle Disease Subunit Vaccine
title_full_unstemmed	Expression, Purification And Quantitation Of Hemagglutinin-Neuraminidase For Dose-Ranging Assessment Of A Recombinant Protein-Based Newcastle Disease Subunit Vaccine
title_sort	expression, purification and quantitation of hemagglutinin-neuraminidase for dose-ranging assessment of a recombinant protein-based newcastle disease subunit vaccine
granting_institution	Universiti Sains Malaysia
granting_department	Institut Penyelidikan Perubatan Molekul
publishDate	2020
url	http://eprints.usm.my/52168/1/LYE%20PING%20YING%20-%20TESIS%20cut.pdf
_version_	1747822138793918464

Expression, Purification And Quantitation Of Hemagglutinin-Neuraminidase For Dose-Ranging Assessment Of A Recombinant Protein-Based Newcastle Disease Subunit Vaccine

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