Purification and analysis of integrity and components of VLPs

Transcription

1 Purification and analysis of integrity and components of VLPs Virus-like particles (VLPs) as vaccines, vectors and adjuvants Fondation Mérieux Conference Center Veyrier du Lac France April 1, 2014 Andris Zeltiņš Latvian Biomedical Research and Study Centre, Ratsupites 1, Riga, LV-1067, Latvia

2 History: First VLPs at Latvian Biomedical Research & Study Centre HBcAg VLPs (1987) Qβ VLPs (1993) Bacteriophage fr VLPs (1987) G. Borisova, M. Bundule, E. Grinstein, D. Dreilina, A. Dreimane, J. Kalis, T. Kozlovskaya, V. Loseva, V. Ose, P. Pumpen, P. Pushko, D. Snikere, E. Stankevica, V. Tsibinogin, E.J. Gren. Mol. Gen. (Life Sci. Adv.) 1987, 6: Kozlovska TM, Cielens I, Dreilina D, Dislers A, Baumanis V, Ose V, Pumpens P. Gene. 1993; 137(1):

3 Examples of VLPs constructed at Latvian Biomedical research & study centre AP205 CB5 fr GA PP7 Qβ SP P. pastoris P. pastoris S. cerevisiae P. pastoris E.coli P. pastoris E.coli Bacterial VLPs ApMV RBDV CCMV RYMV (T3) RYMV(T1) RGMoV CfMV E.coli E.coli E.coli E.coli E.coli P. pastoris E.coli Plant VLPs ACLSV ASPV ASGV PVX PVY PVM E.coli E.coli E. coli E.coli E.coli E.coli Mammalian VLPs HBcAg E.coli / yeasts / mammalian cells HaPyV E.coli / Drosophila S2

4 Gene synthesis Hospital / veterinary specimen Environmental samples Purification and analysis of integrity and components of VLPs How to obtain new VLPs? Bacteria Yeasts Viral nucleic acid CP cdna cloning Insect cells Mammalian cells - Cell disruption - Early identification - Purification - Components of VLPs - Stability - Storage VLPs-? direct CP cdna cloning Plants Cell-free synthesis

5 Buffer systems preserving the integrity of VLPs - Ionic strength (high/low salt content); - Metal ions (e.g. Ca 2+, Mg 2+ ); - Chelating aģents (e.g. EDTA); - Reducing agents (e.g. DTT, mercaptoethanol); - Protease inhibitors (e.g., EDTA, PMSF, protease inhibition «cocktails»); - Urea; - Nucleases (DNAses, RNAses, nuclease benzonase etc.); - Detergents (e.g. TX-100, Tween 20); - Precipitation with ammonium sulfate or PEG; - For new VLPs buffer systems derived from isolation protocols of corresponding native viruses ensuring the structural integrity and/or infectivity;

6 Cell disruption Freezing / thawing grinding with Al2O3 French press Ultrasonication focused, non-contact Ultrasonication conventional RYMV-K VLPs RGMoV VLPs RGMoV VLPs RYMV VLPs RYMV-K VLPs? E.coli P.pastoris P.pastoris E.coli Cell disruption have to be efficient and preserve the integrity of VLPs

7 Procedures useful for early detection of VLPs Analytical ultracentrifugation / SDS/PAGE Typical VLPs, no structures in background M S P No VLPs found Visual test S P M Some VLPs found, most CP in aggregates M S P

8 Procedures useful for early detection of VLPs Agarose gels / Crude lysates M A1 A2 A1 A Benzonase treatment M Benzonase treatment M ul After PEG8000 precipitation VLPs A2 VLPs 1 VLPs 2 VLPs 3? Agarose gel analysis can suggest the presence of VLPs in the samples

9 Purification: ultracentrifuge gradient separation M T S1 P1 S2 P M ApMV VLP purification: SW32 rotor (Beckman), rpm, 6 h, discontinuous sucrose gradient (20-60%, 1xPBS, 0.5% TX-100), fraction size 5 ml Second sucrose gradient (10-40%, 1xPBS, 0.5% TX-100), fraction size 4 ml ApMV VLPs

10 Purification: column chromatography Size-exclusion chromatography Ion exchange chromatography RYMV VLPs; Fractogel TMAE, 20 mm Tris/HCl; 0 2M NaCl HBc- E1 M S Fract. No. RYMV VLPs Skrastina D, Petrovskis I, Petraityte R, Sominskaya I, Ose V, Lieknina I, Bogans J, Sasnauskas K, Pumpens P. Clin Vaccine Immunol. 2013, 20 (11): HBc VLPs

11 Characterization of VLPs: mass spectrometry SDS/PAGE Western blot M 0 T 1 M 0 T PVY CP Mw theor = PVY VLPs M GNDTIDAGGSTKKDAKQEQGSIQPNLNKEKEK DVNVGTSGTYTVPRIK... Kalnciema I, Skrastina D, Ose V, Pumpens P, Zeltins A. Mol Biotechnol. 2012; 52(2):

12 Characterization of VLPs: mass spectrometry SDS/PAGE phosphoprotein staining 1 - HBc VLPs E.coli 2 - phosphorylated HBc VLPs P. pastoris Freivalds J, Dislers A, Ose V, Pumpens P, Tars K, Kazaks A. Protein Expr Purif. 2011; 75(2):

13 Characterization of VLPs: dynamic light scattering HBc- E1 HBc VLPs Skrastina D, Petrovskis I, Petraityte R, Sominskaya I, Ose V, Lieknina I, Bogans J, Sasnauskas K, Pumpens P. Clin Vaccine Immunol. 2013, 20(11): RGMoV VLPs (d=28.5 nm) P. pastoris Dynamic light scattering can serve as an alternative tool for EM

14 Characterization of VLPs: 3D crystallographic models Bacteriophage ϕcb5 VLPs ϕcb5 3D structural model VLP crystals VLPs can serve as an alternative source of viral structures for crystallographic studies Plevka P, Kazaks A, Voronkova T, Kotelovica S, Dishlers A, Liljas L, Tars K. J Mol Biol. 2009; 391(3) :

15 Stability studies: Sypro Orange / Real-time PCR (melting point determination) Principle: - Solution containing VLPs is not activating Sypro Orange fluorescence atroom temperature (hydrophobic surfaces are hidden); - Thermal denaturation expose the hydrophobic surfaces of VLPs and cause Sypro Orange binding and fluorescence; - VLP stability curve and its midpoint value (melting temperature) can be obtained by changing the temperature gradually to unfold the protein and measure the change in fluorescence. 15 mm phosphate +50 mm DTT +25 mm EDTA +0.5 M NaCl RGMoV virions 78 o C 76 o C 65 o C 80 o C RGMoV VLPs 74 o C 73 o C 64 o C 70 o C RGMoV virions RGMoV VLPs from P. pastoris

16 Stability studies: storage of VLPs - Optimal VLP concentration; - Salt concentration - Additives (e.g., glycerol, sucrose, trehaloze, sorbitol, polysorbat); - Freezing (-20oC/-80oC) / storage at +4oC Aggregated VLPs after freezing Disassembled VLPs after freezing

17 Characterization of VLPs: introduced epitopes Mass spectrometry Competitive ELISA PVY VLPs PVY-preS1 VLPs PVY CP PVY-preS1 CP M GNDTIDAGGSTKKDAKQEQGSIQPNLNKEKEK DVN... M GNDNPLGFFPDHQLDPAFRANTANPGSGGGGSGGGGSGGGGS TIDAGGSTKKDAKQEQGSIQPNLNKEKEK DVN The surface localization of the pres1 epitope was demonstrated by competitive ELISA. Increasing amounts of competitor antigen (PVY CP pres1; PvyCP,) were added to the pres1 specific mabma18/7 and the binding of antibodies to the immobilized pres1 peptide was measured. Kalnciema I, Skrastina D, Ose V, Pumpens P, Zeltins A. Mol Biotechnol. 2012; 52(2):

18 Characterization of VLPs: nucleic acid content Next-generation sequencing (Ion Torrent system) RGMoV VLPs from P. pastoris Total reads: Identified sequences: RGMoV CP mrna: ppic3.5 ColE1 transcript: P. pastoris Dienoyl-CoA isomerase (mitochondrial): 966 ppic3.5 Km resistance: 563 P. pastoris chromosome 2 UTR: 346 Other (P. pastoris transcripts):

19 Bacteria Typical workflow for purification and characterization of new VLPs Yeasts Insect Cells Mammalian Cells Plants Cell-free synthesis - «Knowledge-based» buffer systems - «Correct» cell disruption procedure - Early identification: - - analytical sucrose gradients - - agarose gel analysis after nuclease treatment - - electron microscopy - Purification: - - sucrose gradients; - - column chromatography - Characterization: - - estimation of VLP concentrations; - - mass spectrometry; - - dynamic light scattering; - - electron microscopy; - - stability studies including storage conditions; - - nucleic acid packaging studies; VLPs

20 Dr. Ina Baļķe Gunta Reseviča Ieva Kalnciema Jeļena Šaripo Vilija Zeltiņa Acknowledgements: Dr. Kaspars Tārs Dr. Andris Kazaks Dr. Ivars Petrovskis Dr. Velta Ose Dr. Dace Skrastiņa Dr. Dāvids Fridmanis Prof. Dr. Pauls Pumpēns Prof. Dr. Martin Bachmann (Zurich)