Antiviral Chemistry & Chemotherapy’s current antiviral agents
Novel Nanotechnology-Based Antiviral Agents · Novel Nanotechnology-Based Antiviral Agents: Janice...
Transcript of Novel Nanotechnology-Based Antiviral Agents · Novel Nanotechnology-Based Antiviral Agents: Janice...
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Silver nanoparticle neutralization of hemorrhagic fever viruses
Novel Nanotechnology-Based Antiviral Agents:
Janice Speshock, Ph.D.Saber Hussain, Ph.D.Applied Biotechnology Branch711th Human Performance WingAir Force Research Laboratory
Cleared for public release 88ABW-2009-4491
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Nanomaterials
• Size (< 100nm)
• Optical (metal & Semiconductors
• Magnetic (metal)
• Surface reactivity
• Catalytic activity (high surface area)
• Bioaffinity
• Surface modification
Unique Properties DOD Applications
•Biosensors
•Antimicrobial Agents
•Munitions
•Propellants
•Coatings
•Smart Suits
Challenges
• Toxicity
• Reproducibility
• Stability of coatings/functional groups
• bioaffinity
• Effects on protein activity
• Effects on gene expression
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Silver Nanoparticles
UNCOATED POLYSACCHARIDE COATED
10 nm
25 nm
12.78 ± 0.13Dr. Steve Oldenburg, NanoComposix
9.48 ± 4.286Dr. Dan Goia, Clarkston University
27.474 ± 9.062Dr. Karl Martin, Novacentrix
25.98 ± 8.38Dr. Dan Goia, Clarkston University
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Hemorrhagic Fever Viruses
Arenaviridae• South american HFV,
Lassa Fever, LCMV• Enveloped, RNA viruses• No effective therapies• Candid#1 vaccine• 5-35% fatality rate
Filoviridae• Ebola and Marburg• Enveloped, RNA viruses• No effective therapies• Vaccine in Phase I trials• Up to 90% fatality rate
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Tacaribe Virus
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• New World (Tacaribe) Complex– Junin, Machupo, Guanarito, and
Sabia
• Tacaribe virus is a biochemically andserologically close relative of theCDC category A arenaviruses buthas a low pathogenic potential forhumans
• Experimentally:– Cytopathic effect in vero cells
– lethal meningoencephalitis in mice
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Arenavirus Experimental Setup
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0 hour
1 h.p.i
Vero cells
2 h.p.iConfocal Microscopy
Cell surface expression
4 h.p.iConfocal MicroscopyVirus internalization
4 d.p.iqRT-PCR
8 d.p.iHarvest Progeny VirusTCID50 determination
12 h.p.iTEM
Virus internalization
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TCRV Progeny Virus Production
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Cell Surface TCRV Expression
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Negative Control 10 nm 50 µg/ml 25 nm 50 µg/ml
PositiveControl 10 nm 10 µg/ml 25 nm 10 µg/ml
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Dynamic Light Scattering
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TCRV Internalization into Vero Cells
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Untreated TCRV TCRV + 10nm Ag TCRV + 25nm Ag TCRV + 25nm Ag
• Ag-NP-treated TCRV is internalized into infected Vero cells
• Ag-NPs and TCRV interact inside the cell lysosomes
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TCRV Internalization into Vero Cells
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• Ag-NPs facilitate uptake of TCRV into Vero cells
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Nucleoprotein RNA Expression
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Mechanism of Ag-NP Inhibition
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v
vRNA
mRNA
viral protein synthesis
packaging
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Filovirus
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• qRT-PCR detection of internalized eVLPs using Gp as a marker
• Confocal Microscopy of eVLP cell surface binding
• Confocal Microscopy of eVLP internalization
• Cathepsin B and L activity in Vero cells.
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Ebola Virus-Like Particles
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Cell Surface eVLP Expression
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10nm PS-coated Ag 10µg/ml
10nm PS-coated Ag 50µg/ml
25nm PS-coated Ag 10µg/ml
25nm PS-coated Ag 50µg/ml
eVLPs(positive control)
10nm uncoated Ag 10µg/ml
10nm uncoated Ag 50µg/ml
25nm uncoated Ag 10µg/ml
25nm uncoated Ag 50µg/ml
Vero cells(negative control)
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eVLP Internalization into Vero Cells
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Negative Control 10nm 10μg 10nm 50μg
Positive Control 25nm 10μg 25nm 50μg
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Cathepsin Activity
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Cathepsin BL. Jayashankar, Acharya Nagarjuna University , Guntur
• Bulk and Nano Silver have been shown to inhibit enzyme activity.
• Silver binds readily to thiolgroups.
• Cathepsin B has been shown to have an essential role in Ebola virus replication.
• Cathepsin L has an accessory role in Ebola virus replication.
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Cathepsin Inhibition
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10nm Ag 10µg/ml 10nm Ag 50µg/ml 25nm Ag 10µg/ml 25nm Ag 50µg/mlCathepsin B(positive control)
10nm Ag 10µg/ml 10nm Ag 50µg/ml 25nm Ag 10µg/ml 25nm Ag 50µg/mlCathepsin L(positive control)
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Conclusions
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• Ag-NPs neutralize TCRV infection• Decrease in S segment gene expression• Decrease in progeny virus production
• Ag-NPs do not prevent the internalization of TCRV• Ag-NPs and TCRV interact inside the cell• Mechanism of inhibition occurs between endocytosis
and vRNA gene production• Ag-NPs have a similar effect on eVLPs• Ag-NPs decrease cathepsin activity
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Acknowledgements
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• Dr. Schlager (AFRL/RHPB) and Col. Reilly (AFRL/RH)• Funding: DTRA (proposal #4.10036_07_AHB_B) & JSTO/DTRA – NRC
Postdoctoral Fellowship Program (contract #F49620-02-C-0015)
AFRL/RHPB BIN Group
Nanoparticles•Dr. Karl Martin (Novacentrix, Austin, TX)•Dr. Steven Oldenburg (NanoComposix, San Diego, CA)•Dr. Dan Goia (Clarkson University, Center for Advanced Materials Processing, Potsdam, NY)
Ebola virus-like particles•Dr. Kelly Warfield (USAMRIID)Dr. Laura Braydich-Stolle, Craig Murdock, Eric Szymanski
Dr. Amanda Schrand – not pictured
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