Lowcostproductionoftetravalentdenguevaccineintobaccochloroplasts

EvenS.Riiser1,IngridHoltsmark1,HegeS.Steen1,Sathyamangalam Swaminathan2,Navin Khanna2,RalphBock3,JihongL.Clarke1

Norwegian Institute for Agricultural and Environmental Research

Introduction
Dengue viruses (Flavivirus) are mosquito-borne human pathogens with a worldwide prevalence. There are four antigenically-related dengue virus serotypes, DEN-1 to DEN-4, which cause serious problems of morbidity and mortality. Dengue is emerging rapidly as one of the most important public health problems in countries of the AsiaPacific region with nearly 1.8 billion people in the region at risk, compared to an estimated total of 2.5 billion globally (fig. 1A) (WHO 2009). The disease has resulted in widespread social and economic problems, especially among the poor, who are the most vulnerable group. There is currently no vaccine to prevent dengue virus infection (Etemad et al. 2008) making it difficult to control and manage the disease, although considerable efforts have been made, including vector control, sanctions, law enforcement and public education (www.who.int). A DEN vaccine should be tetravalent, as immunity to a single serotype does not offer crossprotection against the other serotypes (Hombach et al. 2005, Etemad et al. 2008). Such a tetravalent vaccine candidate has already been expressed successfully in the yeast Pichia pastoris (Etemad et al. 2008). In this study, we aim to develop a similar envelope domain III (EDIII)-based tetravalent antigen, as well as the individual EDIIIs as monovalent candidates. The antigens will be expressed in tobacco chloroplasts, aiming for a cost-effective and safe production system by joint efforts of IndoNorwegian bilateral collaboration.

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Ongoing and future work

Several versions of the monovalent and tetravalent constructs (fig. 2A) have been synthesized, with sequences codon optimized for their expression in the chloroplast genome of tobacco. All variants of the codon optimized EDIII-constructs have been cloned into suitable vectors, and most constructs have been transformed into tobacco. Positive transformants have been identified by PCR-screening (fig. 3A) and transferred to soil (fig. 3B). Next, homoplasmy will be assessed by Southern blotting, before recombinant protein will be extracted from leaf tissue, and the level of expression (% total soluble protein) determined. The immunological properties of the recombinant vaccine candidate will subsequently be tested.

Figure1.A)Countries/areasatriskofdenguetransmission,2008(WHO2008) B)Aedesaegyptii,thedenguevectormosquito(tropisme.wordpress.com)

Project objectives
1. Development and characterization of new experimental mono- and tetravalent vaccine candidates against dengue, an important mosquito-borne viral disease, by expressing the host cell receptor binding dengue EDIII of all four dengue virus serotypes. 2. Engineering of tobacco chloroplasts to facilitate a cost-effective and safe production system for dengue and other human and animal vaccines.

Methodology
EDIII-encoding sequences corresponding to all four DEN virus serotypes will be fused, generating expression vectors for the production of tetravalent dengue vaccine (fig. 2A). Several variants of this construct, as well as monovalent versions, will be transformed into tobacco chloroplasts using the biolistic transformation method, and subsequent selection will give raise to homoplasmic plants. The recombinant antigen (fig. 2B) will then be purified and subjected to immunological testing. Tobacco will be used because it is a non-food and non-feed crop with excellent biomass. It is an ideal choice for the production of vaccine antigens because of its relative tractability to genetic manipulation and an impending need to explore alternative uses. Furthermore, chloroplast transformation will be used to express EDIII antigens due to low production costs, low risk of contamination with human pathogens, and easy upscaling capacity.
Figure3.(A)PCRamplificationofa326bp EDIIIvectorspecificfragmentidentifies severalpositivetransformants.The300bp fragmentrepresentsannonspecificPCR productalsopresentintheWTplants. (B)Positivetransformantshavebeen transferredtosoil.PhotosbyEvenSannesRiiser,2011.

Conclusions
The use of plants for vaccine production offers several advantages. Unlike the bacterial and mammalian expression systems, plants are ideal for the production of clean and safe vaccine antigens free of contaminants. Plant systems are more economic as they can be produced on a larger scale than industrial systems. There is also minimized risk of contamination from potential human pathogens as plants are not hosts for human infectious agents. The EDIII-based recombinant protein is a promising candidate for the development of a safe, efficacious, and inexpensive tetravalent dengue vaccine.

Acknowledgements
A) B)
EDIII1 EDIII2 EDIII3 EDIII4 6xHis

EDIII-2 EDIII-1 EDIII-3

Figure2.Schematic drawingof(A)the synthesizedDNA constructand(B)the recombinantpolyprotein. (EvenSannesRiiserand NavinKhanna)

We thank ICGEB colleagues for their support in providing the necessary information, including their previous work on the dengue virus and the antigen sequences. This poster represents an IndoNorwegian bilateral project on dengue vaccine financed jointly by the GLOBVAC program of the Research Council of Norway for Bioforsk and BOKU, and the Indian Department of Biotechnology for ICGEB. References
Etemad B, Batra G, Raut R, Dahiya S, Khanam S, Swaminathan S, Khanna N (2008) Am. J. Trop. Med. Hyg. 79(3) 353-363. Hombach J, Barrett AD, Cardosa MJ, Deubel V, Guzman M, Kurane I, RoehrigInnis BL JT, Sabchareon A, Kieny MP (2005). Vaccine 23: 26892695.

EDIII-4

www.bioforsk.no

E-mail: even.s.riiser@bioforsk.no
1NorwegianInstituteforAgriculturalandEnvironmentalResearch(Bioforsk),Hgskoleveien7,N1432s,Norway 2TheInternationalCentreforGeneticEngineeringandBiotechnology(ICGEB),110067NewDelhi,India 3UniversityofNaturalResources&AppliedLifeSciences(BOKU),1180Vienna,Austria

Bioforsk Plant Health and Plant Protection Hgskolveien 7, 1432 s Norway