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PCR-generated linear DNA fragments utilized as a hantavirus DNA vaccine
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
Swedish Defence Research Agency, Division of CBRN Defence and Security, SE-901 82 Umeå, Sweden.
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2002 (English)In: Vaccine, ISSN 0264-410X, E-ISSN 1873-2518, Vol. 20, no 27-28, 3379-3388 p.Article in journal (Refereed) Published
Abstract [en]

The field of DNA vaccines has grown rapidly, and since most such vaccines involve the inoculation of large circular DNA molecules previously propagated in bacteria, several inconveniences (e.g. the presence of antibiotic resistance genes, impurities from bacterial cultures or inefficient uptake of the large and bulky plasmid DNA molecules to the nucleus) are debated. In this study, we have explored the possibility of using smaller and more flexible PCR-generated linear DNA fragments instead. Phosphorothioate (PTO)-modified primers were used successfully to protect the PCR-generated DNA fragments from exonuclease degradation, and by using a nuclear localization signal-peptide to target the linear DNA to the nucleus the immune response against the encoded antigen was further improved. This approach was tested in cell culture using a sensitive reporter system and in vivo with DNA encoding the amino-terminus of the Puumala hantavirus nucleocapsid protein. Our results indicate that linear DNA fragments have a great potential as a genetic vaccine and phosphorothioate modification in combination with a nuclear localization signal peptide increase the stability and targets the linear DNA molecules to the nucleus resulting in an improved biological response examined both in vitro and in vivo.

Place, publisher, year, edition, pages
2002. Vol. 20, no 27-28, 3379-3388 p.
URN: urn:nbn:se:umu:diva-4575DOI: 10.1016/S0264-410X(02)00265-7PubMedID: 12213408OAI: diva2:143730
Available from: 2005-05-09 Created: 2005-05-09 Last updated: 2012-03-09Bibliographically approved
In thesis
1. Implications of Local Puumala Hantavirus Genetics and Epidemiology for Diagnostics and Vaccine Development
Open this publication in new window or tab >>Implications of Local Puumala Hantavirus Genetics and Epidemiology for Diagnostics and Vaccine Development
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Puumala viruses, a member of the Hantavirus genus in the Bunyaviridae family, are enveloped by a lipid bilayer and possesses a tripartite single stranded RNA genome with negative polarity. The hantaviruses encode four proteins: a nucleocapsid protein (N), two membrane spanning glycoproteins (GN and GC) and a RNA dependent RNA polymerase (RdRp). Hantaviruses cause two forms of diseases, hemorrhagic fever with renal syndrome (HFRS) in Europe and Asia, and hantavirus pulmonary syndrome (HPS) in the Americas. The hantaviruses are mainly rodent borne, and humans are mostly infected by inhalation of aerosolized rodent secrete. Human Puumala virus infection results in nephropathia epidemica (NE), a mild haemorrhagic disease.

It is of importance to have a good understanding of the epidemiology and genetics of these viruses for the development of new diagnostic methods and for future vaccine development.

In this thesis we determined the complete viral genome sequence and characterized the structural proteins based on studies of expression and glycosylation patterns, for a unique human virus isolate; performed a genomic analysis of local Puumala viruses and their individual rodent host, Clethrionomys glareolus, from six different locations was performed. It was seen that the virus genetic variation between different locations could be stable over relatively large distances while there could be large variation over a short distance. For the bank voles no such variation could be seen; developed and evaluated Genetic vaccines, based on PCR-generated linear DNA. We showed that it was important to protect these fragments against nuclease degradation at that attachment of a nuclear localization signal peptide further improved the immune response. We also designed, fabricated and evaluated a 2000 probe cDNA-microarray for identification and differentiation of hantaviruses. The chips was based on 12 different strains of six hantaviruses and could differentiate between both different hantaviruses and strains within one hantavirus serotype.

Place, publisher, year, edition, pages
Umeå: Klinisk mikrobiologi, 2005. 97 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 964
Communicable diseases, hantavirus, Puumala virus, nephropathia epidemica, genome sequence, glycosylation, linear DNA vaccine, microarray, identification, genetic variability, Infektionssjukdomar
National Category
Infectious Medicine
Research subject
Medical Virology
urn:nbn:se:umu:diva-532 (URN)91-7305-878-5 (ISBN)
Public defence
2005-06-03, A05, 6A, Umeå Universitet, Umeå, 09:00 (English)
Available from: 2005-05-09 Created: 2005-05-09 Last updated: 2009-11-16Bibliographically approved

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