Interactions of viral particles and osmolytes for manufacturing, detection, and inactivationCARYN L. HELDT

Department of Chemical Engineering, Michigan Technological UniversityArab-American Frontiers of Science, Engineering, and Medicine Symposium

Rabat, Morocco November 2017

Background & Motivation Results & Discussion

ReferencesReferences 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: 83-90o 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

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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 Sensitiveo Inexpensive o Portableo Detect virus non-specifically

Advantages:

To detect virus using AuNPs aggregation induced by osmolytes

AuNPs aggregation

Osmolyteaggregation Virus detection

AbstractIn this age of modern medicine, viral diseases continue to take millions of lives. Ourlab uses osmolytes to manipulate viral particle associations. Osmolytes are naturallyoccurring compounds that regulate osmotic pressure by controlling the structure ofwater molecules. Osmolytes can manipulate water molecules that surround viralparticles. By understanding the interaction of water around large, hydrophobic viralparticles, we can engineer methods to purify, detect and inactivate viral particles.

Osmolytes

SaltWaterOsmolyte

Normal cell Osmolyte action

Nucleus Nucleus Nucleus

Osmolytes bind water and maintain cell volume

HeldtBioseparationsLaboratory

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 shapeo Highly hydrophobico Dehydration causes

aggregation

o Flexible shapeo Less hydrophobico 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 MackChair in Bioengineering, and Michigan Technological University.

Manufacturing

Detection

Inactivation

Model Virus TitrationPorcine parvovirus Sindbis virus

Abbreviation PPV SINVCapsid Non-enveloped Enveloped

Nucleic Acid ssDNA ssRNASize (nm) 18-26 48-52

pI ~5.5 ~4.2Model 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) Transfusionictvdb.orgictvdb.org

High Virus Concentration

FormazanMTT

Low Virus Concentration

Infectious virus detected with an MTT assayo Set up the same as a TCID50o Put cells in a 96-well plateo Put virus sample in the left wellso Serially dilute virus across the plateo After virus infection, detect viable cells with the

MTT dye

Gold nanoparticles (AuNPs)

Size

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Size

nanocomposix.com

2 nm 150 nm

With an increase in AuNP sizeo peak broadeningo red-shift in UV spectrum

With an increase in AuNP sizeo obvious color changeo 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

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Antiviral Activity Post Infection

Viable virus production inside and outside cells

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*Significant difference between osmolyte and PPV (p<0.05)

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3.0 M Mag Sulf 1.0 M Ala1.0 M Man Water

Batch virus flocculation

10 mL 10 mL 10 mL

Mannitol Mannitol

Filtrate 15 mL

Virus Mannitol

Retentate 1 Retentate 2 Retentate 3

VirusOsmolyte

Filtrate 25 mL

Filtrate 35 mL

5 mL5 mL

5 mL5 mL

Semi-continuous virus flocculation

0.5 µm100 nm0.5 µm

Mannitol Water

Filter pore size

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Mannitol Water

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Pore Size

6 logs

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Diafiltration Purity

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Pore Size

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Enveloped virusDiafiltration

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SINV starting concentration0.1 µm filter6 logs 9 logs 10 logs

Non-enveloped virus

6 logs

Osmolytes disrupt capsid assembly

Tafur et al (2013) Antiviral ResearchTime (hours)

PP

V/D

API

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GlyTMAOPPVPBS

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*Significant difference between osmolyte and PPV (p<0.10)

Combined

PPVCapsid

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.

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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.

ControlScreening

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Concentration (log10(pM)) Concentration (log10(pM))𝑨𝑨𝑨𝑨 =

𝞴𝞴 𝒎𝒎𝒎𝒎𝒎𝒎 + 𝟔𝟔𝟔𝟔𝟔𝟔𝒎𝒎

𝞴𝞴 𝒎𝒎𝒎𝒎𝒎𝒎

∆𝑨𝑨𝑨𝑨 = 𝑨𝑨𝑨𝑨𝑩𝑩𝑩𝑩𝑩𝑩𝑩𝑩𝑩𝑩𝑩𝑩 𝒕𝒕𝑩𝑩𝑩𝑩𝒎𝒎𝒕𝒕𝒎𝒎𝑩𝑩𝟔𝟔𝒕𝒕 − 𝑨𝑨𝑨𝑨𝑨𝑨𝑩𝑩𝒕𝒕𝑩𝑩𝑩𝑩 𝒕𝒕𝑩𝑩𝑩𝑩𝒎𝒎𝒎𝒎𝑩𝑩𝟔𝟔𝒕𝒕

∆∆𝑨𝑨𝑨𝑨 = ∆𝑨𝑨𝑨𝑨𝑺𝑺𝒎𝒎𝑺𝑺𝒕𝒕 − ∆𝑨𝑨𝑨𝑨𝑶𝑶𝑶𝑶𝒎𝒎𝑩𝑩𝑺𝑺𝑶𝑶𝒕𝒕𝑩𝑩