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WELCOME

Nanoparticles

• Size: 1 to 100 nm

• Organic and inorganic in nature

• Metalic, liposomes and dendrimers etc.

• Used as carrier molecules

• Problems- toxic, non-degradable and non-specific

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Viral nanoparticles - a versatile nanomachine

Saurav Saha(2014-11-106)

Centre for Plant Biotechnology and Molecular BiologyCollege of Horticulture

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Outline

• Introduction to virus

• Virus as a nanomachine

• Development of viral nanoparticles

• Applications

• Challenges

• Recent achievements

• Summary

• Conclusion 4

Introduction to virus

Naturally occurring biomolecule

Size- 15 to 2000 nm

Rod-like or spherical in shape

Capsid- outer protein coat

Genomic material- DNA or RNA

Deliver genome in host cells

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(Grasso and Santi, 2010 )

• High strength of capsid protein

• Polyvalent and self-assembly process

• Monodisperse structure

• Mass production of viruses

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(Alexander et al., 2013)

Virus as a nanomachine

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(Kristopher et al., 2015)

…virus as a nanomachine

Development of viral nanoparticle

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Virus

Genetic engineering Bioconjugation Biomineralization Encapsulation

modifications

• Epitope sequence

• Targeting sequence

• Unnatural amino acid

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(Merzlyak et al., 2008)

Genetic engineering approach

Coating of various substances within another material

Assemble and de-assemble in-vitro

Artificial polymer, enzyme, metallic nanoparticles

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Encapsulation

(Carissa et al., 2010)

pH > 6.5- swells and release RNA molecule

pH decreases- PSS molecule assemble and entrapped

Polystyrene encapsulation

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(Soto et al., 2010)

Biomineralization

Accumulation of minerals in cells and tissues

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(Wang et al., 2012)

Contd…

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BVSE- Biomineralized- based virus shell engineering

CAR- Coxsackie virus and adinovirus receptor (Wang et al., 2012)

Bioconjugation

Two main strategies

Standard bioconjugation chemistries

Copper-catalyzed azide-alkyne cycloaddition

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(Smith et al., 2013)

Standard bioconjugation chemistries

15(Smith et al., 2013)

Azides and alkynes in the presence of copper

Popularly known as click reaction

Conjugation of protein building block

Copper-catalyzed azide-alkyne cycloaddition

16(Smith et al., 2013)

Application of viral nanoparticles

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Viral nanoparticles (VNP)

Targeted drug delivery Vaccines Imaging Plant diseases

management

Targeted drug delivery

Reduce drug toxicity and degradation

Target particular organ

Increase bioavailability and circulation

18( Nicholas et al., 2014)

Anti-cancer drug

Poor selectivity

High cardio toxicity

Doxorubicin (DOX) delivery

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(Zeng et al., 2013)FA- Folic acid

Cytotoxicity of various DOX formulations at different DOX concentration

20( Zeng et al., 2013)

Dox CMV-DOX CMV-FA-DOX0

102030405060708090

Apop

totic

per

cent

age

DOX concentration (5 µg/ml) incubate for 3 hr

Effect of DOX on cardiomyocytes cells

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( Zeng et al., 2013 )

Vaccine Production

Virus like particles (VLPs)

Antigen stability

Potential to carry two or more different antigen (chimera)

22( Kristopher et al., 2015)

Flock house virus VLP production

23( Destito et al., 2009 )

Universal influenza vaccine from VLP

Globally 250,000–500,000 deaths annually

Virus continually evolving

H1, H2, H5, H6, H7, H10, and H11 hemagglutinin subtypes

Universal vaccine are most effective

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(Kang et al., 2009)

.…universal influenza vaccine from VLP

Mice vaccinated with mixture of 1.5g each of H1, H3, H5, and

H7 VLPs

Mice were boosted at 21 days post immunization

After 35 days of immunization

Mice are infected with different strains of virus

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(Kang et al., 2009)

Effect of homologous challenge

26(Kang et al., 2009)

Body

wei

ght (

%)

Surv

ival

(%)

(Days)

H1N1

Effect of heterosubtypic challenge

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Body

wei

ght (

%)

Surv

ival

(%)

Days

H6N1

(Kang et al., 2009)

Days

VLP type Antigen Indication Product name

Status Reference

Non-enveloped

Virus structural protein

HBV GenHevac B® Licensed Soulie et al., 1991

Non-enveloped

Virus structural protein

HPV Cervarix® Licensed Agnandji et al., 2012

Non-enveloped (chimeric)

Parasite protein Malaria RTS,S Phase 1 El-Attar et al.,2009

Enveloped (virosome )

Parasite protein Malaria PEV3 Phase 1/2 Cech et al., 2011

HBV-Hepatitis B virusHPV-Human papilloma virus

Other types of VLP based vaccine

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Vaccine for tumor/ cancer

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Tn glycan over expressed

Low immunogenicity of carbohydrates (Tn)

Induce a strong T cell-dependent immune

Cowpea mosaic virus (CMV) conjugate with Tn glycoprotein

( Miermont et al., 2008 )

day 0 day35 day 35+ GAlNac

day 35 comtrol

0

2000

4000

6000

8000

10000

12000

14000

16000Ti

tre

ELISA titres of anti-Tn conjugate with CMV

30(GAINac- Tn antigen) ( Miermont et al., 2008 )

control IgG control IgM0

2000

4000

6000

8000

10000

12000Ti

tre

Titre of different types antibodies

31( Miermont et al., 2008 )

Visual representation of internal structures

Synthetic dye ,quantum dot and green fluorescent protein

Low sensitive and photo stability

VNP used as carrier for imaging dye

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Imaging

( Yoo et al., 2012)

Intravital vascular imaging

Fd- Florescine dextranA4d- A488 dextranRhl- Rhadomine-leveled

33(Lewis et al., 2006)

Effect of injection of viral nanoparticle based fluorescence dye on mouse

34(Lewis et al., 2006)

In -vivo stability of fluorescent viral nanoparticles

35( Yoo et al., 2012)

Worldwide crop damage - 157 million dollar

Highly toxic contact and fumigant nematicides

Abamectin (Abm)- biologically active compound

Immobile in soil and photo-oxidative in nature

VNPs used as carrier for abamectin

Plant parasitic nematode control

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Red clover necrotic mosaic virus (RCNMV) as VNP

37( Richard et al., 2015)

Performance of VNP loaded abamectin

Time (Hour)

pH 5.2

A- Within 5 hours 95% of the chemical diffuse out

B- Within 5 hours 25% of the chemical diffuse out

38( Richard et al., 2015)

pH 5.2

A B

Effect of VNP loaded abamectin on tomato seedling

39Gall No gall ( Richard et al., 2015)

Recent achievements

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• Herpes virus- genetically modified

• BioVex company in USA

• FDA approval for treating cancer

• Brand name imlygic

Cancer-hunting virus

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(Bell et al., 2015)

Carbon-free hydrogen fuel

Hydrogenase enzyme

Protons (H+) and electrons (e-) into molecules of H2

P22 bacteriophage coat protein to encapsulate

Mixed with Protons and electrons-ferrying molecules

Hydrogen produced inside a virus

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( Robert et al., 2015 )

Challenges

• Purity in compound

• Encapsulate contaminants

• Manufacturing- not scalable or cost-effective

• Structural complexity

• Baculovirus as a vector

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(Crisci et al., 2012 )

Summary

Viral nanoparticles

Features of viral nanoparticles

Modification strategies

Targeted drug delivery, vaccination and imaging

Plant disease control

Major Challenges

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• VNPs used as biological nanocarrier

• Enormous application in biomedical and agriculture

• Modification- chemical and genetic

• Drug, toxin and targeted sequence

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Conclusion

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“Where Nature finishes producing its own species, man begins, using natural things and with the help of this nature, to create an infinity of species”

Leonardo-Da-Vinci

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Thank youThank youThank you