Vaccine Technology

1. Vaccine Technology

2. Vaccine Technology

3. Vaccine Technology

4. Vaccine Technology Strategy Examples of pathogens targeted Recombinant protein production Hepatitis B S Ag, pertussis toxin, Lyme outer surface protein A, CMV gB protein Live relatedrecombinants Dengue genes in yellow fever 17D, parainfluenza 1+2 genes in parainfluenza 3,M. tuberculosis genes in BCG Alpha virus replicons HIV, Hemorrhagic Fevers Replication-defective particles HPV, SARS ‘Naked’ DNA plasmids HIV and many others Prime boost using DNA/vectors HIV, malaria, tuberculosis Reverse vaccinology Meningococcus B Microarrays for virulence genes Mainly bacteria Synthetic peptides Cancer, CTL vaccines Synthetic polysaccharides Hib Reverse genetics Influenza, parainfluenza, RSV

5. Vaccine Technology • Newer Technology: • Mammalian cell culture: live and killed vaccines • Subunit vaccines: • From serum (HBV) or virus / bacterial disruption • Made via recombinant DNA technology • Recombinant vaccine vectors • DNA vaccines

9. Institute ofInfectiousDisease &Molecular Medicine Dept Molecular& Cell Biology Putting Viruses to Work Ed Rybicki Subunit Vaccines Group

10. Viral Vector Systems The Virus as Slave…

11. Baculovirus expression

13. Ways to Express Foreign Proteins in Plants • Make plants transgenic:- stable system BUT- problems with low expression levels- problems expressing multiple Ags • Use transient expression vector:- high-level expression- can express multiple Ags • POTENTIALLY MUCH QUICKER DEVELOPMENT TIME

14. OPEN READING FRAMES tRNA-likestructure subgenomic promoters 5’ 3’ Vector genome 5’ 3’ translation transcriptionfrom RNA(-) leaky stop codon 126 kDa 5’ 3’ 183 kDa replication-associated proteins 30 kDa 17 kDa Coatprotein Movement protein FOREIGNPROTEIN Tobacco mosaic virus vector

15. Subunit Vaccines Group (UCT) • Working on production of vaccines to Human papillomavirus (HPV) types 11 and 16 and Human immunodeficiency virus (HIV) type 1 subtype C in baculovirus and plant transient and transgenic expression systems • Developing Porcine circovirus (PCV) type 1 as a DNA vector for animal and human vaccination • Developing candidate vaccines for Beak and feather disease virus of psittacines

16. Why an HPV Vaccine? “The presence of HPV in virtually all cervical cancers implies the highest worldwide attributable fraction so far reported for a specific cause of any major human cancer.” Prof. Jan M. WalboomersDepartment of PathologyThe Free University of Amsterdam Digene slide

17. UCT baculovirus-produced VLPs

18. Chimaeric L1 VLPs expressing a L2 neutralising epitope • A common-neutralizing Ab epitope for HPVs 16 and 18 is foundbetween aa 108-120 of the HPV 16 minor capsid protein, L2 • The L2 epitope was introduced into 5 regions of the HPV-16 L1 gene by PCR.  

19. C A E F H Monomeric structure of L1 F-G E-F D-E C-D h4 Chen et.al. 2000 A: loop E-F; C: loop D-E; E: region between H4 & H5 helix; F: H4 helix; H: internal loop C-D

20. Only ChiC and E make VLPs

21. V5 E70 U4 9A J4 I23 D9 L2 Chi A + + + + + + + + 174-186 Chi C - - + + + + + + 131-143 Chi E + + + + + + + + 431-443 Chi F + + + + + + + + 414-426 Chi H - - - - + + + + 81-93 L1 + + + + + + + - antibody characterisation of chimaeric VLPs 266-289

23. Processed Gag proteins Unprocessed Pr55 Gag FIRST GENERATION VACCINES BASED ON HIV-1C GAG Gag- p55

24. Baculovirus production of HIV Virus-Like Particles A. Jaffray, E.P. Rybicki.

25. Harvesting VLPs of THMgag from a 30-70% sucrose gradient.

27. Routine VLP Production: • Presently at batch 109! • Regular QC established • Purification process optimised (& troubleshot) • Shaking cultures Sf9 cells established • Ann making Psi-seq GFP-coding RNA to encapsidate: as marker and “filler”

28. Will use PCV vector for: -SAAVI HIV vaccine -HPV vaccines

29. Comparing replicating and non-replicating plasmid vectors: Grttn expression in human embryonic kidney cells pTHPCVgrttn-RepF pTHgrttn (+) pTHPCVgrttn-RepR pTH (-)

31. DNA coding for virus protein Introduce into plant by genetic engineering Produce edible material OR Harvest plants, extract protein DOSE HUMANS Formulate into pills or capsules Plant-Derived Vaccines: OR by plant virus X

32. ADVANTAGES OF ORAL PLANT-PRODUCED VACCINES • Safe - no human pathogens • Multivalent / multicomponent vaccines possible • Needle-free delivery • Production can be “low tech” so can be done in the developing world • Production is CHEAP…. • (Hugh Mason, IBC’s Fifth International Congress on Vaccine Technologies)

33. Baculovirus - produced HIV Gag VLPs 110 - 120 nm diameter Plant - produced HIV Gag VLPs Production of HIV-1C Gag VLPs via TMV vector in N benthamiana

34. TMV COAT PROTEIN FUSIONS Fiona Tanzer Gama Bandawe Ziyaad Valley-Omar COATED TMV PARTICLE C N TMV VIRION To express p24, p17, env, nef, tat and revpeptidesas N- or C-terminal orinternal fusions

35. EM Work TMV+151a epitope (TMV+151a epitope) + anti-V3

36. Expression ofL1 constructs Wild-type Plantised Humanised H16.V5 capture 17.1% TSP H16.J4 capture 650 1.6 GFP capture 4.5% TSP 600 1.4 550 14.9% TSP 500 1.2 450 GFP (OD492nm) 1 400 350 mg of L1 per kg plant material 0.8 300 250 0.6 200 0.4 150 100 0.2 50 0 0 pTRA- pTRAERH- pTRACTP- pTRA- pTRAERH- pTRACTP- pTRA-HL1 pTRAERH- pTRACTP- pTRACTP- Plant SAL1 SAL1 SAL1 SYNL1 SYNL1 SYNL1 HL1 HL1 HL1ΔC22 control

37. Secreted alkaline phosphatase (SEAP) assay

38. A tobacco field… L1 expression has gone from: Ridiculous (~4 ug/kg) to Sublime (~250 mg/kg) …and the implications of that are…

39. … with a final yield of 25 mg/kg of vaccine protein (=10% oftotal L1) = 500 grams of vaccine protein, or 5 million doses/ha at 100ug dose =worth of US$500 million retail for present HPV VLP vaccine candidate…

40. Vac- cines

41. The End…