- Molecular biology of positive-stranded RNA viruses
- Immunobiology of flavivirus infections
Department of Medical Microbiology
Conserved flavivirus RNA sequences and structures and their function in the the virus life cycle.
Identification and functional analysis of viral and host proteins that are involved in the replication of flaviviruses.
Strategies to express heterologous genes from yellow fever virus (YFV) based vectors to develop effective bivalent YFV-17D based vaccines.
Characterization of the innate and adaptive immune responses against YFV-17D and heterologous genes expressed via YFV-17D in relevant model systems and humans.
Yellow fever virus is the type member of the genus Flavivirus, a group of mainly arthropod-borne RNA viruses. The virus is endemic in the central regions of Africa and South America, where it primarily infects non-human primates. Occasionally YFV infected Aedes and Haemogogus mosquitoes transmit the virus to human populations, which can result in epidemics with mortality rates that vary depending on the YFV strain involved. It is estimated that there are annually 200.000 cases of YFV including 30.000 deaths. There is no specific treatment for yellow fever. Vaccination with the attenuated YFV-17D strain is the single most important measure for protection against infection. The vaccine is safe, without significant side effects and is highly effective; protecting 95% of the vaccines with in one week after vaccination with a single dose and probably resulting in a life-long protection.
The YFV genome is a positive-stranded RNA molecule of 11.8 kb, with a 5’ cap structure and none-polyadenylated 3’ terminus. Translation of the single ORF encode on the vial genome results in the production of a precursor protein that is cleaved by host and viral proteases to produce the mature viral proteins. Processing of the N-terminal one-third of this polyprotein results in the structural proteins C, prM and E. Proteolytic cleavage of the remainder of the precursor protein yields the viral non-structural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. The 5’ and 3’ none-translated regions (NTR) of the viral genome contain various RNA elements that are often well conserved within the flaviviruses. Especially the 3’ NTR is predicted to fold into a complex RNA structure involving several conserved nucleotide sequences, conserved stem-loop structures and RNA pseudoknots.
We were the first to report the construction of a full-length YFV infectious cDNA that was stably maintained in E.coli. Currently, we are using this clone to study the replication of YFV in cell culture and to develop attenuated YFV-based vaccines that not only protect against YFV but also from other infectious diseases that are endemic in the regions in which people are at risk for yellow fever.
Our current research on the molecular biology of flaviviruses focuses on RNA pseudoknots that have been predicted to be present within the YFV 3’-NTR. Using a combination of chemical RNA structure probing and mutational analysis we have been able to confirm the formation of these tertiary RNA structures and are currently studying their function in the YFV live cycle.
The research into YFV-vectored recombinant vaccines focuses on recombinant YFV-17D viruses that express Lassa virus (LAV) antigens. Despite initial success to protect Guinea pigs from a lethal dose of LAV after immunization using an YFV recombinant expressing LAV GP1 and GP2, subsequent studies showed this recombinant YFV-17D virus to be genetically instable. We are currently focussing in YFV recombinants that express either GP1 or GP2. We will use a combination of these two viruses in our attempts to produce a genetically stable and safe vaccine that does not only protect people in Africa from YFV but also from Lassa fever. This work is done in collaboration with Dr I.S. Lukashevich.
In collaboration with Dr. L.G. Visser, we are studying the immune response of humans upon vaccination with YFV-17D. These studies are aimed at determining the qualitative and quantitative characteristics in the adaptive immune response when using alternative strategies to administer the vaccine and to compare the effectiveness of the vaccine in various age groups.
Peter Bredenbeek did his undergraduate work at the “Nieuwe Leraren Opleiding” (NLO) in Utrecht to become a teacher in Biology and Chemistry. As part of this education he was involved in studies determining key plant species to monitor the succession of plant communities in the Dutch National Park “De Weerribben”. After graduation from the NLO, Peter studied at Utrecht University majoring in animal physiology (phosphorylase activation and activity in Locusta migratoria) and virology (Coronavirus replication) and receiving his Msc in 1984. Peter did his Ph.D. in the Department of Veterinary Virology at Utrecht University under supervision of Prof. Dr. B.A.M. van der Zeijst and Prof. Dr. W.J.M. Spaan, studying various aspects of coronavirus replication. After defending his thesis in January 1990, Peter continued his career as a post-doctoral fellow in the laboratory of Prof. Dr. C.M. Rice at that time at Washington University in St. Louis, developing Sindbis virus-based expression systems and studying yellow fever virus replication.
In November 1992 Peter returned to the Netherlands to join the newly established molecular virology research group (then headed by Prof. Dr. W.J.M. Spaan) at the Department of Medical Microbiology of Leiden University Medical Center. The initial years in Leiden were spent in fruitful collaboration with (now Dr.) Rene Rijnbrand on studying IRES mediated translation initiation in hepatitis C – and pestiviruses. However, gradually Peter's interest in 'classical flaviviruses' took over. In recent work he is using yellow fever virus as a model system to study the role of conserved RNA sequences in flavivirus replication and is exploring strategies to make YFV-17D based bivalent vaccines.
Peter also is coordinator of student projects and other teaching activities of the research sections of the Department of Medical Microbiology. He is involved in the block Molecular Biology for LUMC students in Biomedical Sciences and co-coordinator of the Frontiers of Science course Molecular Virology.
Roukens AH, Soonawala D, Joosten SA, de Visser AW, Jiang X, Dirksen K, de Gruijter M, van Dissel JT, Bredenbeek PJ, Visser LG. Elderly subjects have a delayed antibody response and prolonged viraemia following yellow fever vaccination: a prospective controlled cohort study. PLoS One. 2011; 6:e27753.
Yi Z, Sperzel L, Nürnberger C, Bredenbeek PJ, Lubick KJ, Best SM, Stoyanov CT, Law LM, Yuan Z, Rice CM, MacDonald MR. Identification and characterization of the host protein DNAJC14 as a broadly active flavivirus replication modulator. PLoS Pathog. 2011; 7:e1001255.
Jiang X, Dalebout TJ, Bredenbeek PJ, Carrion R Jr, Brasky K, Patterson J, Goicochea M, Bryant J, Salvato MS, Lukashevich IS. Yellow fever 17D-vectored vaccines expressing Lassa virus GP1 and GP2 glycoproteins provide protection against fatal disease in guinea pigs. Vaccine. 2011; 29:1248-57.
Silva PA, Pereira CF, Dalebout TJ, Spaan WJ, Bredenbeek PJ. An RNA pseudoknot is required for production of yellow fever virus subgenomic RNA by the host nuclease XRN1. J Virol. 2010; 84:11395-406.
Roukens AH, Vossen AC, Bredenbeek PJ, van Dissel JT, Visser LG. Intradermally administered yellow fever vaccine at reduced dose induces a protective immune response: a randomized controlled non-inferiority trial. PLoS One. 2008; 3:e1993.
Silva PA, Molenkamp R, Dalebout TJ, Charlier N, Neyts JH, Spaan WJM, Bredenbeek PJ. Conservation of the pentanucleotide motif at the top of the yellow fever virus 17D 3'stem-loop structure is not required for replication. J Gen Virol. 2007; 88:1738-1747.
Bredenbeek PJ, Molenkamp R, Spaan WJM, Deubel V, Marianneau P, Salvato MS, Moshkoff D, Zapata J, Tikhonov I, Patterson J, Carrion R, Ticer A, Brasky K, Lukashevich IS. A recombinant yellow fever 17D vaccine expressing Lassa virus glycoproteins. Virology. 2006; 345:299-304.
Charlier N, Molenkamp R, Leyssen P, Paeshuyse J, Drosten C, Panning M, de Clercq E, Bredenbeek PJ, Neyts J. Exchanging the yellow fever virus envelope proteins with modoc virus prM and E proteins results in a chimeric virus that is neuroinvasive in SCID mice. J Virol. 2004; 78:7418-7426.
Bredenbeek PJ, Kooi EA, Lindenbach B, Huijkman N, Rice CM, Spaan WJM. A stable full-length yellow fever virus cDNA clone and the role of conserved RNA elements in flavivirus replication. J Gen Virol. 2003; 84:1261-1268.
Molenkamp R, Kooi EA, Lucassen MA, Greve S, Thijssen JCP, Spaan WJM, Bredenbeek PJ. Yellow fever virus replicons as an expression system for hepatitis C virus structural proteins. J Virol. 2003; 77:1644-1648.
Leids Universitair Medisch Centrum
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