Interactions of viral particles and osmolytes for manufacturing, detection, and inactivation CARYN L. HELDT Department of Chemical Engineering, Michigan Technological University Arab-American Frontiers of Science, Engineering, and Medicine Symposium Rabat, Morocco November 2017 Background & Motivation Results & Discussion References References by author: o Tafur, M. F., Vijayaragavan, K. S. and Heldt, C. L. (2013). Reduction of Porcine Parvovirus Infectivity in the Presence of Protecting Osmolytes. Antiviral Research 99, 27-33. o Gencoglu, M.F., Pearson, E. and Heldt, C.L. (2014). Porcine parvovirus flocculation and removal in the presence of osmolytes. Journal of Biotechnology 186: 83-90 o Gencoglu, M. F. and Heldt, C. L. (2015) Enveloped Virus Flocculation and Removal in Osmolyte Solutions. Journal of Biotechnology. 206, 8-11. TEM image of synthesized AuNPs 0 0.5 1 1.5 2 400 500 600 700 Normalized Absorbance Wavelength (nm) 519 nm 525 nm 0 4 8 12 16 20 1 10 100 1000 10000 Intensity (%) Size (nm) 15 nm 21 nm 41 nm UV-Vis spectra of AuNPs Size of synthesized AuNPs Limit of detection Synthesized AuNPs PPV coated AuNPs BSA coated AuNPs 50 nm o Quick o Sensitive o Inexpensive o Portable o Detect virus non-specifically Advantages: To detect virus using AuNPs aggregation induced by osmolytes AuNPs aggregation Osmolyte aggregation Virus detection Abstract In this age of modern medicine, viral diseases continue to take millions of lives. Our lab uses osmolytes to manipulate viral particle associations. Osmolytes are naturally occurring compounds that regulate osmotic pressure by controlling the structure of water molecules. Osmolytes can manipulate water molecules that surround viral particles. By understanding the interaction of water around large, hydrophobic viral particles, we can engineer methods to purify, detect and inactivate viral particles. Osmolytes Salt Water Osmolyte Normal cell Osmolyte action Nucleus Nucleus Nucleus Osmolytes bind water and maintain cell volume Heldt Bioseparations Laboratory Osmolytes reduce protein aggregation http://www.chemistry.emory.edu Amyloid proteins spontaneously aggregate and cause diseases, like Alzheimer’s disease Protecting osmolytes slow aggregation Denaturing osmolytes increase aggregation Nayak et al. 2009, Biotech Prog N-oxides Glycine TMAO Urea Amino Acids Sugars Mannitol N-oxides > Sugars > Amino Acids > Denaturing High ΔG of transfer AA in Water AA in Osmolyte ΔG Amino acids interact with water and not osmolytes Denaturant Low ΔG of transfer Street et al. 2006, PNAS Virus interaction with osmolytes Water Protein Osmolyte Virus Add Osmolyte Proteins Virus Add Osmolyte o Rigid shape o Highly hydrophobic o Dehydration causes aggregation o Flexible shape o Less hydrophobic o Dehydration causes compaction and stabilization Acknowledgements This work has been supported by the National Science Foundation (CBET-1159425, CBET-1125585, and CAREER-1451959), EMD Millipore, the James and Lorna Mack Chair in Bioengineering, and Michigan Technological University. Manufacturing Detection Inactivation Model Virus Titration Porcine parvovirus Sindbis virus Abbreviation PPV SINV Capsid Non-enveloped Enveloped Nucleic Acid ssDNA ssRNA Size (nm) 18-26 48-52 pI ~5.5 ~4.2 Model for B-19 human parvovirus, hepatitis A virus, and poliovirus Eastern and western equine encephalitis viruses, hepatitis C Dalrymple et al. (1976) Virology; Miesegaes et al. (2013) Biotechnol. Prog; Simpson et al. (2002) J. Mol. Biol; Stramer et al. (2009) Transfusion ictvdb.org ictvdb.org High Virus Concentration Formazan MTT Low Virus Concentration Infectious virus detected with an MTT assay o Set up the same as a TCID 50 o Put cells in a 96-well plate o Put virus sample in the left wells o Serially dilute virus across the plate o After virus infection, detect viable cells with the MTT dye Gold nanoparticles (AuNPs) Size 400 500 600 700 800 2.0 1.5 1.0 0.5 0 Wavelength (nm) Absorbance (a.u.) 10 nm 20 nm 30 nm 40 nm 50 nm 60 nm 70 nm 80 nm 90 nm 100 nm Size nanocomposix.com 2 nm 150 nm With an increase in AuNP size o peak broadening o red-shift in UV spectrum With an increase in AuNP size o obvious color change o also occurs with aggregation Protein corona stabilizes AuNPs and stops aggregation in salt. (Ho et al. (2015) Analyst) For vaccine manufacturing, osmolytes can be used as virus flocculants by inducing hydrophobic interactions between virus particles that do not occur in most proteins. This allows for the separation of virus from contaminating proteins using a large pore-size membrane. Osmolyte flocculation can purify an enveloped and non-enveloped leading to a cost- effective purification method for a variety of viral products. Osmolytes were explored as a method to inactivate virus. Glycine and TMAO were able to stop cell lysis and the release of infectious virus particles. It was determined that capsid proteins are still produced in an infected cell, but no infectious virus particles can be found. It was concluded that osmolytes, which stabilize proteins, stabilize the individual capsid proteins and interfere with the capsid assembly process. This process could be a new method to inactivate virus particles post-infection. Toxicity Antiviral Activity 0 2 4 6 0.00 0.10 0.20 0.30 Gly TMAO Log reduction value Concentration (M) % Survival of PK-13 cells 0 25 50 75 100 0.00 0.20 0.40 0.60 Gly TMAO Concentration (M) Time post-infection(hours) Log reduction value 0 2 4 6 0 4 8 12 16 20 Gly TMAO 0.2 M osmolyte Antiviral Activity Post Infection Viable virus production inside and outside cells * * 0 2 4 6 8 0 10 20 30 40 Outside Outside Gly Outside TMAO Time (hours) log 10 (MTT 50 /ml) *Significant difference between osmolyte and PPV (p<0.05) * * * 0 2 4 6 8 0 10 20 30 40 Inside Inside Gly Inside TMAO Time (hours) log 10 (MTT 50 /ml) 0 20 40 60 80 100 PPV SINV BSA Lysozyme Percent Removal 3.0 M Mag Sulf 1.0 M Ala 1.0 M Man Water Batch virus flocculation 10 mL 10 mL 10 mL Mannitol Mannitol Filtrate 1 5 mL Virus Mannitol Retentate 1 Retentate 2 Retentate 3 Virus Osmolyte Filtrate 2 5 mL Filtrate 3 5 mL 5 mL 5 mL 5 mL 5 mL Semi-continuous virus flocculation 0.5 µm 100 nm 0.5 µm Mannitol Water Filter pore size 0 20 40 60 80 100 0.1 µm 500 kDa 300 kDa Percent Recovery Mannitol Water * * *p< 0.05 Pore Size 6 logs 0 20 40 60 80 100 Percent Recovery 1st fraction 2nd fraction 3rd fraction 6 logs 9 logs 11 logs PPV starting concentration 0.1 µm filter Diafiltration Purity 0 20 40 60 80 100 0.4 0.6 0.8 1.0 1.2 1.4 15 17 19 21 23 Percent buffer B eluted Absorbance (mAU) Time (Minutes) Before filtration Filtrate Retentate 0.1 µm filter 0 20 40 60 80 100 0.2 µm 0.1 µm 500 kDa Percent Recovery Mannitol Water Filter pore size * * * Pore Size 6 logs E nveloped virus Diafiltration 0 20 40 60 80 100 Percent Recovery 1st fraction 2nd fraction 3rd fraction SINV starting concentration 0.1 µm filter 6 logs 9 logs 10 logs Non-enveloped virus 6 logs Osmolytes disrupt capsid assembly Tafur et al (2013) Antiviral Research Time (hours) PPV/DAPI 0 100 200 300 4 8 12 16 20 24 Gly TMAO PPV PBS * * * *Significant difference between osmolyte and PPV (p<0.10) Combined PPV Capsid DAPI 20 hour time point PBS Control 0.2M TMAO + PPV 0.2M glycine + PPV PPV Control Capsid protein detection in cells Osmolytes aggregation of virus is being used as a virus detection method. The aggregation of virus- coated AuNPs can be detected by the UV spectrum shifts found by the aggregation of AuNPs. We are able to measure a difference between the aggregation of virus-coated and proteins-coated AuNP by comparing the aggregation in salt and osmolyte solutions. -0.1 0.1 0.3 0.5 4 6 8 ΔΔAI 0 0.2 0.4 0.6 0 3 6 ΔΔAI Other references: o Ho, Y. T., B. Poinard, E. L. Yeo and J. C. Kah (2015). An instantaneous colorimetric protein assay based on spontaneous formation of a protein corona on gold nanoparticles. Analyst 140(4): 1026-1036. o Nayak, A., C. C. Lee, G. McRae and G. Belfort (2009). Osmolyte controlled fibrillation kinetics of insulin: New insight into fibrillation using the preferential exclusion principle. Biotechnol. Prog. 25(5): 1508-1514. Control Screening 0 0.2 0.4 0.6 0.8 4 6 8 ΔAI PPV-AuNPs Concentration (log 10 (MTT 50 /mL)) 0 0.2 0.4 0.6 0.8 0 3 6 ΔAI BSA-AuNPs PPV-AuNPs Concentration (log 10 (MTT 50 /mL)) Concentration (log 10 (pM)) Concentration (log 10 (pM)) ∆ − ∆∆ ∆ − ∆