Download Dengue Fever – plant derived vaccines and other coping strategies

Dengue Fever – plant derived vaccines and other
coping strategies in a changing climate
Jihong Liu Clarke
Bioforsk- Norwegian Institute for Agricultural and Environmental
Research, Høgskoleveien 7, 1432 Ås, Norway
[email protected]
Dengue fever
(DF) and dengue haemorrhagic
fever (DHF), and their global status
 How
would climate change affect vector
borne diseases such as dengue?
 Coping
strategies: prevention, early warning,
production of effective and affordable vaccines
in plants, vector control, law enforcement,
public education etc.
 Summary and future work
Aedes aegypti
http://www.who.int 2009




Infected female mosquito transmits dengue
Primarily a daytime feeder
Lives around human habitation
Lays eggs in stagnating water
Dengue virus
(+)ssRNA
Family: Flaviviridae
Genus: Flavivirus
Species: Dengue virus
Serotypes: 4 (DEN 1-4)
Source: Dr. Kanna, ICGEB, India
Dengue: Current Global Status
 2.5 billion people
at risk
 50 million DF cases/yr
 500,000 DHF, largely children
 DHF fatality rates 2.5-20%
 No drugs or vaccine
 Included in WHO/TDR
WHO (2009)
Dengue continues to spread
around the globe
Only 9 countries had DHF epidemics before 1970
Exceeded 100 countries in 2009 (www.who.int)
MacKenzie et al., Nature Med., 2004
Dengue continues to spread around the globe
Recently, an outbreak of dengue fever on the island of Pulau Besar outside
Malaysia was reported (www.dagbladet.no/2010/05/24).
Dengue
fever (DF) and dengue haemorrhagic
fever (DHF) and the global status
 How
would climate change affect vector
borne diseases such as dengue?
 Coping
strategies: prevention, early warning,
production of effective and affordable vaccines
in plants, vector control, law enforcement,
public education etc.
 Summary
and future work
May promote “vertical” spread of
dengue to previously unexposed regions
and countries (reaching higher)?
Example: Aedes aegypti, the dengue
causing mosquito, was previously found
normally at a height of not more than
500 metres above sea level. Of late,
these have been sighted at an altitude
of 2200 meters in Darjeeling and 4000
meters in Nepal (Dr. Jai P Narain 2008)
Photo: FreakingNews.com
Global warming may also
contribute to spreading of
dengue “horizontally”?
As a result of increased temperature which is optimum for
mosquito breeding, vector borne diseases will have new
destinations where populations were earlier unexposed to such
diseases. We are already seeing such cases in Europe.
“Chikungunya (CHIKU, a viral disease spreading by
mosquitoes) resurfaced in India after 31 years in 2006 and
has been reported for the first time from Italy (South Asia
Voice, 2008, Vol 1, p 67).
Coping strategies:
 Prevention
 Vaccination (vaccine is not available)
 Vector control
 Law enforcement
 Public education etc.
Status of dengue vaccine?
No licensed vaccine at present
 Effective vaccine must be tetravalent (ADE)
against all the 4 serotypes (DEN1-4)
 Effective, safe and affordable vaccine is
absolutely needed for controlling dengue

Vaccination is considered to be the most efficient and cost-effective
means for health intervention to combat infectious diseases.
Can plants be used as factories for low cost
production of vaccines?
Production cost using
green leaves
(chloroplast
engineering) is about 1%
of that via traditional
cell cultures
Plants- Green Factories
Plants cells are eukaryotic, like human cells. They can
assemble, modify and secrete complex proteins.
Minimized risk of contamination from human
pathogens as plants are not hosts for the pathogens
Easy scaling up and low production cost (as no initial
investment is required)
No need for field cultivation. Adequate production
scale can be reached in a confined greenhouse.
Plants- Green Factories
(Continued)
Plants are ideal bioreactors for the
production of orally administered
vaccines, eliminating the need for
expensive fermentation,
purification, cold storage and
sterile delivery (Daniell et al. 2009)
Three plant-based production systems
Nulear transformation
Chloroplast engineering
Transient system
Mitochondria
Nucleus
Source: Klemsdal 2008 with modification
Vaccine antigens expressed in plants
Number of
antigens
by 2009
Expression system
Expression level
35
Nuclear
transformation
0 -3.0 %
29
Chloroplast
transformation
0.25-40%
25
Transient system
400 mg kg-1 FW
Daniell et al. 2009. Trends in Plant Science
Progress of plant derived vaccines

DowAgro Newcastle disease vaccine, approved Feb 2006

Therapeutical vaccine for non-Hodgkin‘s lymphoma in Phase I
clinical trial

IFN-α2b to combat hepatitis C has reached Phase II clinical
trial (www.biolex.com).

Human glucocerebrosidase against Gaucher’s disease has
entered Phase III clinical trial
Source: Daniell et al. 2009
Multigene
Engineering
Gene
Containment
High level
expression
Advantages and limitations
of chloroplast
transformation system
No Vector
Sequences
No Gene
Silencing
No Position
Effect
No glycoproteins
expressed in cp
Only successful
in a few crops,
no monocots
Daniell 2006
GLOBVAC: Indo-Norwegian joint project
Expression and evaluation of envelope domain IIIbased experimental tetravalent dengue vaccine
using a tobacco chloroplast expression system
Institutions responsible for the project:
Bioforsk- Norwegian Institute for Agricultural and Environmental
Research, Norway.
ICGEB -The International Centre for Genetic Engineering and
Biotechnology, India
Partner: BOKU-University of Natural Resources & Applied Life Sciences, Austria
Tobacco, an ideal plant factory
Non food, non feed
Objectives
Development and characterization of a new
experimental tetravalent vaccine candidate against
dengue by expressing the host cell receptor binding
dengue envelope domain III (EDIII) of all four dengue
virus serotypes
Engineering of tobacco chloroplasts to facilitate a costeffective production system for the experimental dengue
vaccine which is safe, efficacious and affordable.
Domain III of DEN Envelope Protein
Cell, 2002
 Host cell receptor binding site
 Accessible on virion surface
 Serotype-specific neutralizing epitope
 mAb- efficient virus blockers
 Highly stable
Recombinant domain III protein
can block DEN infection to cells
Independently folding domain
amenable to tetravalent design
Vector construction
DEN-1
DEN-3
DEN-4
Tetravalent candidate was successfully expressed in the yeast P. pastoris
and showed promising immunological response in mice (Etemad et al.
2008).
A
Chloroplast
Transformation Vectors
Single gene:
Cp DNA
Cp DNA
P
T P
Marker
RBS
RBS
Multiple genes:
T
X
RBS
Cp DNA
Cp DNA
P
Marker
X
Y
Z
T
B
With antibiotic
Gene delivery
Selection
Regenerated plantlets
Regeneration
C
Eukaryotic
Nuclear genome
Nucleus
Prokaryotic
Plastid/chloroplast
Mitochondria
A heteroplasmic diploid plant
cell (First round of selection)
A homoplasmic diploid plant cell
(Second round of selection)
Daniell 2006
Photo: Even Sannes Riiser
Summary
 Understanding the impact of climate change on vector borne
diseases such as dengue is essential for their effective
management in the future
 Multi- disciplinary coping strategy including vector control,
production of effective, safe and affordable vaccine etc. is
required for controlling DF and DHF
 Plant-based vaccine production systems, in particular the
chloroplast engineering-based system, could facilitate the
production of a wide array of vaccines in a safe and easy-toscale-up manner with low production cost
 The limitations and challenges of the chloroplast poduction
system must be overcome to advance the technology
Future work
 The expression and functionality of the tobacco chloroplastderived tetravalent dengue vaccine will be investigated
 The economic and social benefits of the chloroplast derived
dengue vaccine will be evaluated
 Expression of the DEN 1-4 antigens in an edible crop (e.g.
broccoli) to produce oral dengue vaccine, further reducing
the production costs and making the vaccine more accessible
and affordable
Acknowledgements
Bioforsk
Even Sannes Riiser
Sonja S Klemsdal
Nagothu Udaya Sekhar
Rebekka Øvstegård
Sissel Haugslien
Research Council of Norway
and DBT in India
ICGEB
Navin Khanna
Sathyamangalam Swaminathan
Vanga Siva Reddy
Sadhu Leelavathi
GLOBVAC
BOKU
Andreas Lössl