Production of dengue virus envelope protein domain III-based antigens in tobacco chloroplasts using inducible and constitutive expression systems.
Dengue fever is a disease in many parts of the tropics and subtropics and about half the world's population is at risk of infection according to the World Health Organization. Dengue is caused by any of the four related dengue virus serotypes DEN-1, -2, -3 and -4, which are transmitted to people by Aedes aegypti mosquitoes. Currently there is only one vaccine (Dengvaxia(®)) available (limited to a few countries) on the market since 2015 after half a century's intensive efforts. Affordable and accessible vaccines against dengue are hence still urgently needed. The dengue envelop protein domain III (EDIII), which is capable of eliciting serotype-specific neutralizing antibodies, has become the focus for subunit vaccine development. To contribute to the development of an accessible and affordable dengue vaccine, in the current study we have used plant-based vaccine production systems to generate a dengue subunit vaccine candidate in tobacco. Chloroplast genome engineering was applied to express serotype-specific recombinant EDIII proteins in tobacco chloroplasts using both constitutive and ethanol-inducible expression systems. Expression of a tetravalent antigen fusion construct combining EDIII polypeptides from all four serotypes was also attempted. Transplastomic EDIII-expressing tobacco lines were obtained and homoplasmy was verified by Southern blot analysis. Northern blot analyses showed expression of EDIII antigen-encoding genes. EDIII protein accumulation levels varied for the different recombinant EDIII proteins and the different expression systems, and reached between 0.8 and 1.6 % of total cellular protein. Our study demonstrates the suitability of the chloroplast compartment as a production site for an EDIII-based vaccine candidate against dengue fever and presents a Gateway(®) plastid transformation vector for inducible transgene expression.
NIBIO-Norwegian Institute of Bioeconomy Research, P.O. Box 115, 1431, Ås, Norway.|BOKU-University of Natural Resources and Life Sciences, Gregor-Mendel-Straße 33, 1180, Vienna, Austria.
Johanna
J
Gottschamel
BOKU-University of Natural Resources and Life Sciences, Gregor-Mendel-Straße 33, 1180, Vienna, Austria.
Andreas
A
Lössl
Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476, Potsdam-Golm, Germany.
Stephanie
S
Ruf
NIBIO-Norwegian Institute of Bioeconomy Research, P.O. Box 115, 1431, Ås, Norway.
Yanliang
Y
Wang
Norwegian University of Life Sciences, Ås, Norway.
Morten
M
Skaugen
Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476, Potsdam-Golm, Germany. rbock@mpimp-golm.mpg.de.
Ralph
R
Bock
NIBIO-Norwegian Institute of Bioeconomy Research, P.O. Box 115, 1431, Ås, Norway. jihong.liu-clarke@nibio.no.
Jihong Liu
http://orcid.org/0000-0002-4924-0406
JL
Clarke
D000595:Amino Acid Sequence; D000956:Antigens, Viral / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D002736:Chloroplasts / Q000235:genetics*; D003716:Dengue Virus / Q000276:immunology*; D000431:Ethanol / Q000494:pharmacology; D005821:Genetic Techniques; D005822:Genetic Vectors / Q000378:metabolism; D030821:Plants, Genetically Modified; D000072417:Protein Domains; D011994:Recombinant Proteins / Q000378:metabolism; D012038:Regeneration; D014026:Nicotiana / Q000235:genetics*; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000378:metabolism
D000956:Antigens, Viral; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D000431:Ethanol
Chloroplast transformation; Dengue virus; EDIII antigen; Plant produced vaccine; Tobacco