Effect of Early Secreted Antigen Target-6 Gene of Mycobacterium Tuberculosis as Genetic Adjuvant for Avian Influenza Virus DNA Vaccine in Chickens

Influenza virus, belongs to the family Orthomyxoviridae and genus Influenza virus A, causes major disease problems and serious economical losses in poultry industry. Highly pathogenic avian influenza H5N1 subtype which is associated with acute infection with high morbidity and mortality in susceptib...

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Main Author:	Oveissi, Sara
Format:	Thesis
Language:	English English
Published:	2009
Subjects:	Influenza viruses Tuberculosis
Online Access:	http://psasir.upm.edu.my/id/eprint/7249/1/IB_2009_4a.pdf
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spelling	my-upm-ir.72492013-06-17T01:48:57Z Effect of Early Secreted Antigen Target-6 Gene of Mycobacterium Tuberculosis as Genetic Adjuvant for Avian Influenza Virus DNA Vaccine in Chickens 2009 Oveissi, Sara Influenza virus, belongs to the family Orthomyxoviridae and genus Influenza virus A, causes major disease problems and serious economical losses in poultry industry. Highly pathogenic avian influenza H5N1 subtype which is associated with acute infection with high morbidity and mortality in susceptible birds, is still enzootic in poultry in Asia as well as European and African countries. The virus may also possess serious threat to the emergence of influenza pandemic in humans. Vaccination is one of the biosafety measures which has the greatest impact on improving global health and preventing morbidity and mortality due to avian influenza (AI) infection. The explosion of knowledge in molecular immunology has paved radical developments in vaccine technology. Immunization with DNA vaccines and genetic adjuvants as immunostimulators is an attractive approach in the development of future generations of vaccines and adjuvants. The viral envelope proteins, hemagglutinin (HA or H) and neuraminidase (NA or N), have been shown to play key roles in triggering protective immune responses against AI infection. Meanwhile, nucleocapsid protein (NP) may play a central role in cross protection between AI virus serotypes. The Mycobacterium tuberculosis Early Secreted Antigenic Target-6 (ESAT-6) antigen has been shown to elicit both humoral and cellular immunity, thus it has an ability to act as a genetic adjuvant. This study examined the ability of ESAT-6 to modulate antibody response against H5 following vaccination with DNA vaccine in chickens. In order to study the immunological properties of AIV DNA vaccines, several recombinant plasmids pcDNA3.1/H5, pcDNA3.1/N1, pcDNA3.1/NP, pcDNA3.1/H5-ESAT6, pcDNA3.1/N1-ESAT6 and pcDNA3.1/NP-ESAT6 were constructed. The recombinant plasmid constructs were confirmed by restriction enzymes and sequence analyses. The expression of genes of interest in cell culture was confirmed by immunofluorescence test and Western blot analysis. The immunogenicity of the DNA vaccine pcDNA3.1./H5 with and without the presence of ESAT-6 in specific-pathogen-free (SPF) chicks was determined. Sera obtained from the chickens immunized with pcDNA3.1/H5 and pcDNA3.1/H5-ESAT6 demonstrated viral neutralizing activities based on haemagglutination inhibition (HI) test. The sera collected from chicks immunized with pcDNA3.1/H5-ESAT6 have higher HI titer compared to the group which was immunized with pcDNA3.1/H5. However, the increase in HI titer at different post immunization days between these groups was not statistically significant. When the tissue samples from the chest muscle of injection site and spleen from chickens immunized with the DNA vaccine were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR), all the samples were positive for H5 specific transcripts. In summary, the current study delineated that the constructed recombinant plasmids were transcriptionally active in the in vivo chicken model and DNA immunization in SPF chicks with pcDNA3.1/H5 and pcDNA3.1/H5-ESAT6 produced humoral immune response. In conclusion, future studies are required to explore the role of ESAT-6 gene of Mycobacterium tuberculosis as an effective genetic adjuvant for H5 DNA vaccine in chickens. Influenza viruses Tuberculosis 2009 Thesis http://psasir.upm.edu.my/id/eprint/7249/ http://psasir.upm.edu.my/id/eprint/7249/1/IB_2009_4a.pdf application/pdf en public masters Universiti Putra Malaysia Influenza viruses Tuberculosis Institute of Bioscience English
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English English
topic	Influenza viruses Tuberculosis
spellingShingle	Influenza viruses Tuberculosis Oveissi, Sara Effect of Early Secreted Antigen Target-6 Gene of Mycobacterium Tuberculosis as Genetic Adjuvant for Avian Influenza Virus DNA Vaccine in Chickens
description	Influenza virus, belongs to the family Orthomyxoviridae and genus Influenza virus A, causes major disease problems and serious economical losses in poultry industry. Highly pathogenic avian influenza H5N1 subtype which is associated with acute infection with high morbidity and mortality in susceptible birds, is still enzootic in poultry in Asia as well as European and African countries. The virus may also possess serious threat to the emergence of influenza pandemic in humans. Vaccination is one of the biosafety measures which has the greatest impact on improving global health and preventing morbidity and mortality due to avian influenza (AI) infection. The explosion of knowledge in molecular immunology has paved radical developments in vaccine technology. Immunization with DNA vaccines and genetic adjuvants as immunostimulators is an attractive approach in the development of future generations of vaccines and adjuvants. The viral envelope proteins, hemagglutinin (HA or H) and neuraminidase (NA or N), have been shown to play key roles in triggering protective immune responses against AI infection. Meanwhile, nucleocapsid protein (NP) may play a central role in cross protection between AI virus serotypes. The Mycobacterium tuberculosis Early Secreted Antigenic Target-6 (ESAT-6) antigen has been shown to elicit both humoral and cellular immunity, thus it has an ability to act as a genetic adjuvant. This study examined the ability of ESAT-6 to modulate antibody response against H5 following vaccination with DNA vaccine in chickens. In order to study the immunological properties of AIV DNA vaccines, several recombinant plasmids pcDNA3.1/H5, pcDNA3.1/N1, pcDNA3.1/NP, pcDNA3.1/H5-ESAT6, pcDNA3.1/N1-ESAT6 and pcDNA3.1/NP-ESAT6 were constructed. The recombinant plasmid constructs were confirmed by restriction enzymes and sequence analyses. The expression of genes of interest in cell culture was confirmed by immunofluorescence test and Western blot analysis. The immunogenicity of the DNA vaccine pcDNA3.1./H5 with and without the presence of ESAT-6 in specific-pathogen-free (SPF) chicks was determined. Sera obtained from the chickens immunized with pcDNA3.1/H5 and pcDNA3.1/H5-ESAT6 demonstrated viral neutralizing activities based on haemagglutination inhibition (HI) test. The sera collected from chicks immunized with pcDNA3.1/H5-ESAT6 have higher HI titer compared to the group which was immunized with pcDNA3.1/H5. However, the increase in HI titer at different post immunization days between these groups was not statistically significant. When the tissue samples from the chest muscle of injection site and spleen from chickens immunized with the DNA vaccine were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR), all the samples were positive for H5 specific transcripts. In summary, the current study delineated that the constructed recombinant plasmids were transcriptionally active in the in vivo chicken model and DNA immunization in SPF chicks with pcDNA3.1/H5 and pcDNA3.1/H5-ESAT6 produced humoral immune response. In conclusion, future studies are required to explore the role of ESAT-6 gene of Mycobacterium tuberculosis as an effective genetic adjuvant for H5 DNA vaccine in chickens.
format	Thesis
qualification_level	Master's degree
author	Oveissi, Sara
author_facet	Oveissi, Sara
author_sort	Oveissi, Sara
title	Effect of Early Secreted Antigen Target-6 Gene of Mycobacterium Tuberculosis as Genetic Adjuvant for Avian Influenza Virus DNA Vaccine in Chickens
title_short	Effect of Early Secreted Antigen Target-6 Gene of Mycobacterium Tuberculosis as Genetic Adjuvant for Avian Influenza Virus DNA Vaccine in Chickens
title_full	Effect of Early Secreted Antigen Target-6 Gene of Mycobacterium Tuberculosis as Genetic Adjuvant for Avian Influenza Virus DNA Vaccine in Chickens
title_fullStr	Effect of Early Secreted Antigen Target-6 Gene of Mycobacterium Tuberculosis as Genetic Adjuvant for Avian Influenza Virus DNA Vaccine in Chickens
title_full_unstemmed	Effect of Early Secreted Antigen Target-6 Gene of Mycobacterium Tuberculosis as Genetic Adjuvant for Avian Influenza Virus DNA Vaccine in Chickens
title_sort	effect of early secreted antigen target-6 gene of mycobacterium tuberculosis as genetic adjuvant for avian influenza virus dna vaccine in chickens
granting_institution	Universiti Putra Malaysia
granting_department	Institute of Bioscience
publishDate	2009
url	http://psasir.upm.edu.my/id/eprint/7249/1/IB_2009_4a.pdf
_version_	1747810680550981632

Effect of Early Secreted Antigen Target-6 Gene of Mycobacterium Tuberculosis as Genetic Adjuvant for Avian Influenza Virus DNA Vaccine in Chickens

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