Expression of ETEC fimbrial antigen CS5 in live attenuated Shigella live vectors
Prosjekt
- Prosjektnummer
- 911997
- Ansvarlig person
- Kurt Hanevik
- Institusjon
- Helse Bergen HF
- Prosjektkategori
- Utenlandsstipend
- Helsekategori
- Infection
- Forskningsaktivitet
- 3. Prevention
Rapporter
This project was conducted during my postdoctoral period abroad from January 2015 to 15th January 2016.During the postdoc period abroad I worked in the laboratory of Professor Eileen Barry. The main aim of the work in this lab is to generate a Shigella vaccine that also protects against ETEC, by making the live attenuated Shigella flexneri 2a strain also express important ETEC colonization factors. By working in this project I acquired skills in cloning, sequencing, primer design, autotransporter constructs, western blots, protein expression and purification, fimbrial extraction, electron microscopy, site directed mutagenesis, guinea pig experiments, and grant application writing. I also attended a very good Vaccinology course that was given at this institution during spring 2015.
I successfully expressed ETEC CS5 fimbriae on three plasmids in DH5a and in the Shigella 1208S vaccine strain. Also, I expressed the two protein subunits of the CS5 fimbriae separately in the sat autotransporter originating from UPEC, and I cloned this into the 1208S vaccine strain on a pBAD plasmid. The immunogenicities of these construct were tested a guinea pig model.
A series of attempts were successfully made to shorten the CS5 and the more well-known ETEC CFA/I fimbriae to increase the relative amount of fimbrial tip adhesin proteins compared to structural proteins by altering a stem-loop region by site directed mutagensis. The results were evaluated by electron microscopy imaging and measurements. Some remaining work adding qPCR and protein blots to confirm these data will be performed at the laboratory after I left. This work will be written up and published by myself.
During my stay I also visited the Bill and Melinda Gates foundation, Seattle in March due to a joint funding application with researches at the Center for Vaccine Development. In October I attended the annual meeting of the annual meeting of the American Society of Tropical Medicine and Hygiene, Philadelphia where I presented my postdoc work on post-giardiasis sequels. In December I visited the Naval Medical Research Center in Silver Spring, Maryland to discuss expression of ETEC adhesion proteins.
Receipts and travel related expenses overview was delivered to Medisinsk avdeling, HUS in April 2016.
Dissemination
Posters, that will become manuscripts for peer-reviewed publications:
Cunningham AL, Hanevik K, Barry EM. Creation and Characterization of Live Attenuated Diarrheal Disease Vaccines Against ETEC and Shigella. Annual Conference of the U.S.-Japan Panel on Cholera & Other Bacterial Enteric Infections. January 11-15, 2016 in North Bethesda, Maryland (USA). The work is likely to result in a co-authored paper.
Hanevik K, Grassel C, Barry E. Increasing anti-adhesin immune responses by modifying fimbrial gene
stem-loop structure in live attenuated Shigella/ETEC vaccines. ASTMH, Atlanta, Nov 2016. This work is in preparation ofr a first-author publication, but awaits some further animal experiments to be performed by staff in Eileen Barrys lab.
My one year postdoc period abroad was done at the Center for Vaccine Development, University of Maryland, Baltimore, USA from 29th Dec 2014 to 14th Jan 2016.During this period I worked in the laboratory of professor Eileen Barry. The main aim of the work in this lab is to generate a Shigella vaccine that also protects against ETEC, by making the live attenuated Shigella strain also express important colonization actors of ETEC. By working in this project I acquired skills in cloning, sequencing, primer design, autotransporter constructs, western blots, protein expression and purification, fimbrial extraction, electron microscopy, site directed mutagenesis, guinea pig experiments, and grant application writing. I also attended a very good Vaccinology course that was given at this institution during spring 2015.
I successfully expressed ETEC CS5 fimbriae on three plasmids in DH5 and in the Shigella 1208S vaccine strain. Also, I expressed the two protein subunits of the CS5 fimbriae separately in the sat autoransporter from UPEC and cloned this into the 1208S vaccine strain. The immunogenicities of these construct were tested a guinea pig model. This work was presented together with other ETEC fimbrial expression work in a poster at the US-Japan Conference on Cholera & other Bacteria Enteric Infections in January 2014. The work is likely to result in a paper with myself as co-author or first author.
Also a series of attempts were successfully made to shorten the CS5 and CFA/I fimbriae to increase the relative amount of fimbrial tip adhesin proteins by altering the CS5 operon by site directed mutagensis. The results were evaluated by electron microscopy imaging and measurements. Some remaining work, adding qPCR and protein blots to strengthen these initial data, will be performed at the laboratory after I left. The work is likely to result in a paper with myself as first author.
During my stay I also visited the Bill and Melinda Gates foundation in March due to a joint funding application with researches at the Center for Vaccine Development. In October I attended the annual meeting of the American Society of Tropical Medicine and Hygiene where I presented my postdoc work on post-giardiasis sequels. In December I visited the Naval Medical Research Center in Silver Spring, Maryland to discuss expression of ETEC adhesion proteins.
Deltagere
- Kurt Hanevik Postdoktor
eRapport er utarbeidet av Sølvi Lerfald og Reidar Thorstensen, Regionalt kompetansesenter for klinisk forskning, Helse Vest RHF, og videreutvikles av de fire RHF-ene i fellesskap, med støtte fra Helse Vest IKT
Alle henvendelser rettes til Faglig rapportering, Helse Vest