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SUBUNIT VACCINES GROUP

SUBUNIT VACCINES GROUP. Inst of Infectious Disease and Molecular Medicine and Dept Molecular and Cell Biology University of Cape Town Inga Becker/Hitzeroth. PRF. Papillomaviruses. Why are they interesting? What does the virus look like? What disease do they cause?

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SUBUNIT VACCINES GROUP

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  1. SUBUNIT VACCINES GROUP Inst of Infectious Disease and Molecular Medicineand DeptMolecular and Cell Biology University of Cape Town Inga Becker/Hitzeroth PRF

  2. Papillomaviruses • Why are they interesting? • What does the virus look like? • What disease do they cause? • Warts • Cervical cancer • How do you catch genital HPV? • Strategies to prevent infections

  3. PV structure 600Å diameter with 72 pentameric capsomeres in a T=7 icosahedral lattice Capsomeres contain 5 monomers of L1 (360) L2 in capsid ranges from 11 - 30 or 72 ds DNA virus DNA size 8kb

  4. HPV DNA

  5. HPV genes • L1 major capsid protein • L2 minor capsid protein • E6 and E7oncogene • E7 binds to pRB, acitivates E2F transcription factor get expression of proteins for DNA replication then p53 should send cell into apoptosis, but • E6 targets p53 for proteolytic degradation • E2 and E1 transcription/translation control • E4 and E5

  6. Infectious cycle • Infect basal epithelial cells • Replicate to about 100 copies, maintained like that for long times • E1 and E2 are essential • HPV replicates in suprabasal cells and are released into environment • E6 and E7 are important in viral replication by interacting with pRB and p53 which control the normal cell cycle • During tumor progression viral DNA often integrates into host chromosome

  7. Papillomaviruses • Species specific • CRPV, BPV, • Human papillomavirus: HPV • Over 100 types • Tissue specific • Epithelial, mucosal

  8. Diseases caused by HPV • Warts and papillomas have been noted since prehistoric times • Infectious nature only discovered later • 1970-80 studied structure of virus, gene functions • Middle 1970’s zur Hausen postulated role of virus in cervical cancer • By 1980’s lots of knowledge about virus had accumulated

  9. HPV types and genes • Mainly HPV 16 DNA was found in cervical cancer biopsies • E6 and E7 were expressed in these cancers • These genes were able to immortalize epithelial cells • These genes were necessary to maintain cancer

  10. Cervical Cancer • Cervical cancer is the 2nd leading cancer amongst women worldwide • 500,000 new cases, 300,000 deaths • >99% related to human papillomavirus (HPV) infection • HPV infect epithelial cells including the genital tract • HPV type 16 related to half of these cases • HPV –18, -45, -31, -33, -52, -58 and -35 together with HPV-16 responsible for 90% of all cervical cancers worldwide

  11. Burden of HPV-related cancers • 500 000 new cases per year • 300 000 deaths of which • 83% occur in developing countries • Cervical cancer accounts for 15% of female cancers • Risk before age of 65 is 1.5% • Developed countries its 3.6% of new cancers with 0.8% risk • Only 55% mortality

  12. Age-standardised (World) incidence rates of cervical cancer 2002

  13. Mostly squamous cell carcinomasAge-standardised (World) incidence rates of cervical cancer by histological subtype in selected cancer registries circa 1993–1997

  14. HPV types • Important to do worldwide study to find out which type is most common in cancer for development of vaccine • Mainly done by PCR and Hybrid capture assay

  15. Percentages of cervical cancer cases attributed to the most frequent HPV types in all world regions combined

  16. Eight most common HPV types in more than 14,500 cervical cancer cases, by region

  17. Progression of natural infection to cancer

  18. HPV Infection • Infection occurs mostly between age 20-30 • Infection is mostly cleared spontaneously • 90% clear a HPV type in 2 years • Some established infections manifest as microscopic abnormality • Also cleared by host immune system • Viral persistence can lead to cancer

  19. Overview of factors most consistently reported to play a role at different stages in the natural history of HPV and cervical neoplasia

  20. Screening for Cervical Cancer • Why get screened? • Screening was developed to detect abnormal cervical cells in the early stages when it is easy to remove them • Who should be screened? • Women from 25-65 years • Once every 3 years

  21. Abnormal cervical smear • What now? • Most abnormal smear revert back to normal spontaneously • Get re-screened in 3 - 6 month • If progress to abnormal cells • Referred for colposcopy – look at cervix, do biopsy and determine if its CIN3 • Remove abnormal cells: cryotherapy, laser, loop or cone biopsy

  22. HPV screening • Why? • If have no HPV DNA go back to routine screening • If high risk HPV DNA is found and it persist, then follow up is very important • HPV screening not recommended for women under 30

  23. HPV Vaccines • HPV L1 • Form VLPs with or without L2 • Look same as viral particles and can be used as vaccine • Problem: • Very type specific • Only protect against the one L1

  24. Structure of HPV M. Stanley et al. / Vaccine 24S3 (2006) S3/106–S3/113

  25. Properties of the HPV L1 capsid protein L1 protein can self-assemble into Virus-like Particles (VLPs) that are antigenically identical to virions - and are already in Phase III human trials (made via yeast and baculovirus) ProjectedCost: US$40-100 per dose of 40-100ug… Natural capsid:L1+L2+DNA Capsid consisting only of L1

  26. Yeast Cell Culture Insect Cell Culture HPV Vaccines • L1 VLPs made in insect cells – GSK 16 and 18 • Cervarix • i.m. 3 shots 0, 1 and 6 month • VLPs made in yeast – Merck 16,18,11 and 6 • Gardasil • i.m. 3 shots 0, 2 and 6 month

  27. Mechanism of protection • Neutralising antibodies • Antibodies are type specific and not cross-neutralising • Duration of protection • Ab persist 48 month post-vaccination • Who and when to vaccinate • Pre-exposure, pre-puberty

  28. New problems • Who should be vaccinated when? • Childhood vaccination programs are well-structures, well-established and accepted • Vaccination must occur prior to onset of sexual activity • Girls at 9-13 years • Few school-based vaccination programs • Boys as well? • Reduce anogenital warts • Increase herd immunity • Target groups: adolescent/young adult women (perhaps men) • Will HPV included in national immunisation program? • Booster vaccination?

  29. Screening programs • Need to continue • Older women • Infection by other HPV types • Even if HPV vaccine is approved, need awareness for need to vaccinate in professional, public, and political communities

  30. Our Research • Production of VLPs in plants • Easy to make • Cheap • L2 contains cross-protective epitopes • L2 has been shown to stabilize capsid formation and allow formation at a physiological pH Belnap et al. (1996).

  31. Plant Vaccine Group University of Cape Town CRPV L1 as a Transgene: Cottontail rabbit papillomavirus is an excellent model system for HPV vaccines as a challenge system can be used. N tabacum cv. Xanthi was transformed with native gene in pART27.

  32. Plant Vaccine Group University of Cape Town Proof of Concept: CRPV Immunisation Transgenic Extracts: Three New Zealand White rabbits inoculated i.m. with ~40 μg L1protein. Two boosters of ~20μg mixed 1:1 with FIA given. Transient Extracts: About 6 ug L1 extract given i.m./s.c. to three New Zealand White rabbits. Two boosters of 3 ug mixed 1:1 with FIA given Sera tested by ELISA and western blot for type-specific antibodies against baculovirus-derived CRPV VLPs Rabbits were then challenged with live CRPV:inoculated with 1/10 and 1/100 dilutions of virus 2x each

  33. Plant Vaccine Group University of Cape Town Controls: BCG-rota (-ve) and CRPV VLPs (+ve) TMV Tr Results of CRPV Challenge: 1st proof of efficacy of a plant-produced papillomavirus L1 protein vaccine

  34. Plant Vaccine Group University of Cape Town N tabacum-produced HPV-16 VLP TMV-producedHPV-16 VLP Arabidopsis-produced HPV-11 VLPs TMV-produced HPV-11 VLP Historical: HPV L1 Transgenics and transients Transgenics: CRPV, HPV-11 and -16 native L1 VLPs were made in all plants except CRPV-transformed. Capsomers appeared in all plants. A thaliana expression was stable to the 6th generation. Yields: HPV-16 L1: ~4 ug/kg HPV-11 L1 NLS-: ~11 mg/kg in At ~2 in Nt CRPV L1: ~1 mg/kg TMV: CRPV, HPV-11 and -16 native L1 VLPs were made in all plants except CRPV-infected. Capsomers appeared in all plants. TMV-CRPV lost the L1 gene early; TMV-HPV16 also eventually deleted. Yields: HPV-16 L1: ~40 ug/kg HPV-11 L1 NLS-: ~11 mg/kg CRPV L1: <1 mg/kg All protein extracts bound conformation-specific MAbs. All extracts tested for immunogenicity by injection into animals as concentrated extracts; sera reacted in ELISA and western blots with appropriate VLPs / proteins

  35. Plant Vaccine Group University of Cape Town direct injection vacuum infiltration Optimising protein production in plants 1. Select optimal constructs using agroinfiltration - simple and rapid method for evaluating protein expression of numerous Agrobacterium constructs 2. Small OR Large-scale infiltration

  36. Plant Vaccine Group University of Cape Town New Transgenic Expression of HPV-16 L1: Investigation ofcodon optimisation andvector effect /subcellular localisation of:- Nicotiana-optimised L1- human-optimised L1 Transient Expression of HPV-16 L1 via Agrobacterium: Investigation ofcodon optimisation andsubcellular localisation of:- wild-type HPV-16 L1 from a clinical sample- Nicotiana-optimised L1- human-optimised L1

  37. Plant Vaccine Group University of Cape Town Pnos LB SAR npt II SAR pAnos ColE1ori P35SS pTRAkc- rbcs1-cTP LB CHS pTRAc P35SS rbcs1-cTP 7743bp 6153bp pA35S bla ColE1ori CHS chloroplast pA35S cytoplasmic SAR bla SAR RK2ori RB RK2ori RB Pnos SAR npt II pAnos P35SS LB pTRAkc-ERH LPH his 6 7701bp KDEL ColE1ori SAR ER bla RB RK2ori Agrobacterium-delivered expression vectors P35SS CaMV 35S promoter with duplicated transcriptional enhancer CHS - chalcone synthase 5' untranslated region pA35S - CaMV 35S polyadenylation signal SAR - scaffold attachment regions LPH - leader peptide from murine heavy chain of mAb24 Thomas Rademacher, Rainer Fischer (Institute for Molecular Biotechnology, RWTH Aachen University, Germany)

  38. 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

  39. Plant Vaccine Group University of Cape Town humanised -> CP Transgenics: localisation effect in N tabacum cv Petite Havana humanised -> ER pTra-CTP in Petite Havana:maximum of >15% TSP (>500 mg/kg)

  40. Optimisation of material to harvest The yield of L1 in line 9 through the transgenic CP-localising plant as determined by ELISA using J4 (red) and V5 (blue) monoclonal antibodies. Leaf number 1 is the youngest leaf

  41. Purification 160 140 120 100 ug L1 / ml 80 60 40 20 0 Initial plant column Elution 1 Elution 2 Elution 3 Elution 4 Elution 5 material flow through Cation exchange purification of HPV-16 L1 transgenic N. tabacum Petite Havana. Columns represent the L1 protein concentration in µg/ml. The values above the columns represent the total amount of L1 protein in the respective fraction as detected using the V5 (white bars) and J4 (grey bars)

  42. Plant Vaccine Group University of Cape Town Transient Expression of HPV-16 L1 Electron micrographs of H16.V5-immuno-trapped crude N. benthamiana plant extract 6 days post infiltration with Agro carrying chloroplast-targeted human codon optimised HPV-16 L1 Plant extracts of transiently produced VLPs were highly immunogenic – producing high neutralising Ab titres

  43. UCT baculovirus-produced VLPs

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