Download Ongoing and future work Low cost production of tetravalent dengue

Low cost production of tetravalent dengue vaccine in tobacco chloroplasts
Even S. Riiser1, Ingrid Holtsmark1, Hege S. Steen1, Sathyamangalam Swaminathan2, Navin Khanna2, Ralph Bock3, Jihong L. Clarke1
Norwegian Institute for Agricultural and Environmental Research
Introduction
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
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,
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.
Ongoing and future work
B)
A)
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
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.
Figure 1. A) Countries/areas at risk of dengue transmission, 2008 (WHO 2008)
B) Aedes aegyptii, the dengue vector mosquito (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.
Figure 3. (A) PCR amplification of a 326 bp EDIII‐vector‐specific fragment identifies several positive transformants. The 300 bp fragment represents an non‐specific PCR‐
product also present in the WT plants. (B) Positive transformants have been transferred to soil. Photos by Even Sannes Riiser, 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)
EDIII‐1
EDIII‐2
EDIII‐3
B)
EDIII-2
EDIII-1
EDIII-3
EDIII-4
www.bioforsk.no
EDIII‐4
6x His
Figure 2. Schematic drawing of (A) the synthesized DNA construct and (B) the recombinant polyprotein. (Even Sannes Riiser and Navin Khanna)
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: 2689—2695.
E-mail: [email protected]
1Norwegian Institute for Agricultural and Environmental Research (Bioforsk), Høgskoleveien 7, N‐1432 Ås, Norway 2The International Centre for Genetic Engineering and Biotechnology (ICGEB), 110067 New Delhi, India
3University of Natural Resources & Applied Life Sciences (BOKU), 1180 Vienna, Austria
Bioforsk Plant Health and Plant Protection
Høgskolveien 7, 1432 Ås
Norway