1. Plants the modern
vaccines
Presented by Lee-Ann Kara 551033
29th April 2014

2. Introduction
 Vaccines
 What makes an effective vaccine?
 The first Vaccine
1885 –Louis Pasteur
http://www.preparedmind.eu/blog/le-hasard-ne-
favorise-que-les-esprits-prepares-louis-pasteur/
Delivery
systems
Antigen
Immune
potentiators

3.  Traditional methods used to
produce vaccines
Different types of currently available vaccines:
1. Live attenuated vaccines
2. Killed vaccines
3. Purified subunit vaccines
4. Recombinant subunit vaccines.
 Recombinant vaccines
 Advantage*
 Hepatitis B virus
 Limitations*
Conventional vaccines:
Costly
Longer time is needed for it to be produced
Heat sensitive
Prone to microbial contamination http://www.onlinerevu.com/top-
10-best-paid-to-click-sites/

4. Biopharming
Two major products in development:
1. Plant derived antibodies.
2. Plant derived subunit vaccines.
Significance of Plant-derived proteins
First plant derived vaccine

5. Types of plants as biofactories for vaccine antibody
production
• leaf and stem tissues of tobaccos of various species and varieties (eg:
Nicotiana benthamiana; N tabacum)
• Arabidopsis thaliana,
• Alfalfa,
• Spinach,
• Potatoes; of
• Aquatic weeds suchas Lemna spp. (duckweed);
• Beans
• Maize
• Tomatoes
• Strawberries;
• Root vegetables like carrots;
• single-cell cultures of the algae Chlorella and Chlamydomonas;

6. Methodologies of vaccine/antibody production
For protein expression:
1. Nuclear transformation
 Agrobacterium mediated transformation.
 Biolistic bombardment transformation.
2. Chloroplast transformation
 Biolistic bombardment tranformation
3. Transient expression
 Agroinfiltration
 Infection with viral vectors

7. Comparison between protein production systems

8. Benefits of using transgenic plants
for vaccine production
1. Eliminates possibility of infections or innate toxicity
2. Concerns with viral contamination are eliminated
3. Economical means of large-scale production
4. Maximization of protein stability
5. Reduced cost
6. Multicomponent vaccines are possible
7. Oral administration is possible

9. SWOT analysis for PDVs

10. Challenges with PDVs
• Technical challenges
• Economical challenges
• Public perception

11. Developement of plant-
derived vaccines against
cervical cancer

12. Objectives:
Aim
To develop and manufacture a plant-derived vaccine therapeutic
against HPV type 16 for females in developing countries which will
thereby decrease their risk of developing cervical cancer
 To establish a cost-effective expression system for HPV-16 L1
Capsomeres
To express the L1_2xCysM gene that is fused with a adjuvant
LTB in tobacco chloroplasts.

13. Introduction:
Cervical cancer
 ~ 493,000 new cases occur every year
 To date 274,000 deaths occur in the world
 By 2030 it is expected to increase to ~ 410,000
 Developing countries have the highest mortality rate due to
cervical cancer is very high

14. Human Papilloma Virus
 High-risk types of HPV include HPV-16, 18, 31,
33, 45, 52 and 58.
 HPV type 16 and 18
HPV
Circular dsDNA virus
No envelope
L1 protein
Capsid protein self assembles into empty
virus like particles (VLPs)
 Modified HPV-16 L1 gene
Disulphide bonds
http://www.virology.wisc.edu/virusworld/images/hpv-HDblue-green.jpg

15. HPV type 16 will be targeted
Currently available conventional vaccines
 Gardasil and Cervarix
 Limitations to current available prophylactic vaccines
against HPV infections:
• These vaccines target only a limited number of HPV types.
• Expensive due to complex processes of production.
• Separate administration of adjuvant
• Maintenance of cooling chain is costly
Therefore a great demand for cost effective vaccines
for women in poorer countries are needed
(Waheed et al., 2012)

16. Second-generation plant-derived vaccines are the perfect
candidates against HPV type 16 infections.
Why?
Because of their,
1. Reduced costs
2. Their stability
3. Increased immunogenicity and
4. High yield

17. Pentameric capsomeres
Capsomeres are found to be highly immunogenic, compared
to levels of VLPs.
Major advantages of Capsomeres:
I. Thermo-stable
II. Easy to produce
III. Can be expressed in plants
 Modified gene coupled to adjuvants

18. The modified HPV-16 L1 gene (L1_2xCysM) was expressed in tobacco
chloroplasts which led to the formation of only capsomeres.
Tobacco chloroplasts was selected as an expression platform
Reasons for using chloroplasts of
Nicotiana tabacum:
 Tobbaco is a well established host
 Tobacco has high Biomass production capacity
 Very high yield of recombinant protein can be obtained
 Helps assure Biosafety
 Rapid scalability
 reduced risk of contaminating feed and human food chains
http://www.gizmag.com/darpa-flu-vaccine/25966/pictures

19. Methodology
Three vectors were constructed for the transformation of tobacco chloroplasts.
Transformation and regeneration of transformed plants
Confirmation of transgenes integration by polymerase chain reaction
Southern blot analysis

20. Protein extraction
Western blotting
Cesium chloride density gradient centrifugation
Sucrose gradient sedimentation analysis of L1 protein

21. Confirmation of proper conformation of recombinant proteins
GM1-ganglioside binding assay
http://www.intechopen.com/books/transgenic-plants-advances-and-limitations/green-way-of-biomedicine-how-to-force-plants-to-produce-new-important-proteins

22. The advantages of plastid transformation method that has been
used which can be exploited for cost effective production of HPV
vaccines:
 High yield of recombinant protein can be achieved
 Scalability: growth of plants can be scaled up according to the required amount of
protein.
 Biosafety issues can be covered by the application of chloroplast transformation and/or
growing the plants in contained facilities.
 Transgenic plants can be grown at the site where the vaccine is needed.
 Stability: plants-derived vaccines are likely to be more stable.

23. Commercialization plan
 Vaccine: Double- pentameric vaccine
 Feasibility
• High yields of this vaccine can be produced
• Biosafety
• This vaccine is stable
• 1.5 % - 2% of TSP is accumulated
• Affordable
• Scalability
• Highly effective
 Relevance and Importance to society:
• More effective than current available.
• HPV plant vaccines can now be available to people in poorer
countries

24.  Potential Market
• Specifically women in developing countries
 Transfer technology for commercialization
 Capital
• Commercialization??
 Production costs: Approximately 160 million Rand is needed to
produce 1 billion doses of the Double- pentameric HPV vaccines
Figure 1: showing the difference in production costs between PDVs and conventional vaccines

25.  Time Frames
• Estimated that the double-pentameric HPV vaccine will take 2
weeks to 90 days to be produced in a tobacco plant system
• Egg-based vaccines take 150 days or even longer
Figure 2: Timelines of vaccines produced in different systems

26.  Administration
• Subcutaneous injections
 Double-pentameric HPV vaccines has a longer shelf life as
compared to current available HPV vaccines

27. References
Ling H-Y, Pelosi A, Walmsley AM (2010) Current status of plantmade
vaccines for veterinary purposes. Expert Rev Vaccines
9,971–982.
Lossl, A.G., and Waheed, M.T. (2011).Chloroplast derived vaccines against human diseases:
achievemnents, challenges and scopes. Plant Biotech Journal 9, 527-539.
Brodzik R, Spitsin, S., Golovkin, M., Bandurska, K., Portocarrero, C., Okulicz, M., Steplewski,
Z.,Koprowski, H. (2008)Plant-derived EpCAM antigen induces protective anti-cancer
response. Cancer immunol Immunother 57,317-323.
Fernandez-San Millan A, Ortigosa, S. M., Hervas-Stubbs, S., Corral-Martinez, P., Segui-
Simarro, J. M., Gaetan, J., Coursaget, P., Veramendi, J.(2008). Human papillomavirus L1
protein expressed in tobacco chloroplasts self-assembles into virus-like particles that are
highly immunogenic. Plant Biotechnol J 6,427-441.
Varsani A, Williamson, A. L., Stewart, D., Rybicki, E. P.(2006). Transient expression of Human
papillomavirus type 16 L1 protein in Nicotiana benthamiana using an infectious
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28. Conley, A.J., Joensuu, J.J., Richman, A., Menassa, R. (2011). Protein body-inducing
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29. Thank You!