Baculovirus mediated gene expression and its

veterinary applications

Presented by : Dr.Madhuvanti V. Mahajan

M.V.Sc Scholar ABT,1246

BVC,Mumbai

Overview Introduction

Various expression systems

Baculovirus

Viruses as expression systems

Baculovirus as expression systems

Advantages of BEVS

Limitations of BEVS

Applications in veterinary science

Current status of BEVS

Future Prospects

Conclusions

Introduction

The vector designed for the expression of i.e, production of protein specified by the DNA insert, is called as Expression Vector.

Expression system - A system in which a cloned gene can be expressed.

Condt…… The increasing demand for production and

characterization of diverse groups of recombinant proteins necessitates the analysis of several constructs and fusion tags in a variety of expression systems.

For this a number of different types of vector systems have been developed. Viz,

Various Vector Systems……..

Bacterial expression system (e.g. E.coli )

Yeast expression system (e.g S.cervesiae)

Viral expression system (e.g Baculovirus)

Mammalian cell expression system

Various Expression Systems

Expression System Selection • Eukaryote – Large proteins (>100

kD)?

• Prokaryote – Small proteins (<30 kD)?

• Baculovirus or mammalian cell culture

– Glycosylation ?

• E. coli – High yields, low cost?

• Yeast or baculovirus or mammalian cells

– Post-translational modifications?

Historical background The history of the discovery of baculoviruses is intimately related to the development of the silk industry that occurred in China as early as 5000 years ago.

By the 12th century it was established in Italy and Spain and eventually spread to France and England and to Mexico by the 1500s.

Since the 1990s they have been utilized for producing complex eukaryotic proteins in insect cell cultures

More recently baculoviruses can transduce mammalian cells when found that a suitable promoter is present

Viral Classification

Group: Group I (dsDNA)

Family: Baculoviridae

Subfamily : Nucleopolyhedrosis Virus (NPV)

Genera : Alphabaculovirus

Betabaculovirus

Deltabaculovirus Gammabaculovirus

Fig. 1.1 Baculovirus occlusion bodies. Scanning EM by K. Hughes

and R.B.Addison.

Viral Morphology Virions exist in two forms:

1. Polyhedra-derived Virus (PDV)

nucleocapsids packaged into

polyhedra (“occlusion bodies”)

Stable in external environment

2. Budded Virus (BV) nucleocapsids

budded from host cells envelope

Involved in secondary infection Fig : Electron micrograph of ODV

Circular, double stranded DNA genome.

A number of small repeated sequences

known as homologous regions (hrs)

interspersed in the genome.

Hrs enhance early gene transcription

and also to act as origins of replication.

Genome

A diagram of the Eppo MNPV genome map

Species-specific tropisms among the invertebrates with over 600 host species.

Immature (larval) forms of moth species are the most common hosts, but these viruses have also been found infecting sawflies, mosquitoes, and shrimp.

They are not known to replicate in mammalian or other vertebrate animal cells.

Host Range

Replication

A ) Viral infection

• Ingestion

• Uncoating of polyhedra

• Fusion with midgut cells

• Viral replication in nucleus

B ) Secondary infection

• Budded virus is released to infect systemically

Replication

Fig : An general overview of the replication cycle of Baculoviruses.

Replication

Phase

Description

Immediate early

Expression of viral transregulators and genes which do not require transregulators.

Delayed early Expression of genes involved in the replication of the virus and manipulation of the host.

Late Characterised by shutdown of the host cell DNA replication and protein synthesis. BV is produced and disseminates

Very late Virions become occluded in the protein polyhedrin. Viral proteases liquefy the host and degrade the chitinous exoskeleton. Occluded progeny virus is disseminated onto surrounding material for horizontal spread.

Baculovirus as an Expression Vector………..

Baculovirus Expression Vector System

Many non-essential genes

- may be replaced by gene of interest

The resulting recombinant Baculovirus lacks one of nonessential gene (polh, v-cath, chiA etc.) replaced with foreign gene

Powerful viral promoters

- particularly for late (L) and very late (VL) phase genes

History…… • To express recombinant genes controlled by

strong insect-virus promoters in their natural host (insect) cells 1983

• It was first published that recombinant baculoviruses are able to deliver genes into mammalian cells. 1995

• Today this is a well-established and easy to handle system for producing large quantities of recombinant proteins for numerous purposes

• These genes are expressed provided that they are controlled by a promoter which is active in mammalian cells

Autographa californica

The virus was originally isolated from the alfalfa looper.

Is multiple nuclear polyhedrosis virus (AcMNPV), which relies

on the lepidopteran species

a) Spodoptera frugiperda and

b) Trichoplusia ni

Fig :Sf9 cell line

Baculovirus as an Expression Vector

Condt…… The major capsid protein VP39 together with some minor

proteins forms the nucleocapsid, that encloses the DNA with p6.9 protein.

BV acquires its envelope from the cell membrane and requires a glycoprotein, gp64, to be able to spread systemic infection.

This protein forms structures called peplomers on one end of the budded virus particle but is not found on ODV.

• Spodoptera frugiperda (Sf9, Sf21) Fall armyworm Isolated from pupal ovarian tissue Spodoptera frugiperda Trichoplusia ni (High Five) Cabbage looper Isolated from egg cell homogenates Trichoplusia ni

The

baculovirus

expression

vector

system

(BEVS)

Steps in recombinant baculovirus production

Figure…

Gene of Interest

Tn7R p10 Gent+ Tn7L

Gene construct

Gene of Interest

Tn7 R

PpH Tn7 L

pfast Bac with insert

Contd..

128bp 145bp

Mini att Tn7 M 13 forward M 13 reverse

Tn7R GOI Tn7L

Bacmid DNA

Transposed pfast Bac

Steps in recombinant baculovirus production

Transposition occurs between the mini-att Tn7 target site to generate a recombinant bacmid

Helper plasmid which allows to transpose the gene of interest from pfast to bacmid (shuttle vector)

Cloned pfast Bac is transformed in E.coli host strain (DH10Bac) which contains a baculovirus shuttle vector bacmid having a mini-attTn7 target site

Expression cassette in pfast Bac is flanked by left and right arms of Tn7

Clone the gene of interest in pfast Bac donor plasmid

Condt…….

Recombinant bacmid is now ready to transfect to insect cell lines

In condition of transposition then the amplified size will be 2300bp+size of insert

If no transposition, then a region a bacmid alone will amplify to gave product of 300bp

PCR amplification using M-13 Forward and Reverse primers

cells Doublin

g time

Cell appearance Medium Origin Type of

culture

Sf 9 72 hrs Spherical, granular,

regular in size, firm

attachment to surface

TNM-FH IPLBSF-21 cell

lines of the fall

army worm

spodoptera

frugiperda

Grow well

as

monolayer

and

suspension

Sf 21 24 hrs Spherical, granular,

different in size, firm

attachment to surface

TNM-FH IPLBSF-21 cell

lines of the fall

army worm

spodoptera

frugiperda

Grow well

as

monolayer

and

suspension

High-

five

18 hrs Spherical, granular,

regular in size, loose

attachment to surface

Express

five

SFM

Ovarian cells of

cabbage looper

Grow well

as

monolayer,

also as

suspension

Why BEVS ?????????

Comparison

Comparison of Expression Systems (Gene Expression Systems. Using nature for the art of expression (Fernandez, J.M. & Hoeffler, J.P., eds), Academic Press, San Diego, 1999)

Characterstics E.coli Yeast Baculovirus Mammalian cells

Cell growth Rapid Rapid Slow Slow

Complexity of growth medium

Minimum Minimum Complex Complex

Cost of growth medium Low Low High High

Expression level High Low-high Low-high Low-moderate

Extracellular Expression Secretion to periplasm

Secretion to medium

Secretion to medium Secretion to medium

Post translational modifications

Protein folding Refolding usually required

Refolding may be required

Proper folding Proper folding

N-linked glycosylation None High mannose Simple, No Sialic acid Complex

O-linked glycosylation No Yes Yes Yes

Phosphorylation No Yes Yes Yes

Acetylation No Yes Yes Yes

Acylation No Yes Yes Yes

Gamma Carboxylation No No No Yes

Comparison of Expression Systems Characteristics

Adenovirus Retrovirus Lentivirus Adeno associated virus

Baculovirus

Infectivity Infects dividing cells

Infects dividing cells

Infects non dividing cells

Infects both dividing and non-dividing cells

Infects dividing cells

Major applications

Gene Therapy and vaccination

Gene Therapy and vaccination

Gene Delivery (Vectors)

Gene Therapy

Gene therapy and vaccine production

Size 7.5 kb 8kb 8kb 4.5kb >15kb

Mode of action

Does not integrate in host genome

provirus, remains and passed on to the cell progeny

Integrates in host genome

Integrates in host genome

Host Immune Response

Strong Moderate Moderate Mild Mild

Advantages

Post translational modification

s

Promoter of the

polyhedrin gene is very

strong

Could be replaced

with a heterologou

s gene

Capable of producing cytotoxic proteins

Can express large

proteins (>50 kD)

Correct glycosylation & signal

peptide removal

Has chaperonins to help fold

“tough” prtns

Very high yields, cheap

Condt………

Glycosylation in insect cells

,different from vertebrate cells,

a problem for therapeutic

proteins.

large fraction RP poorly

processed and accumulates aggregates.

Discontinuous expression

Inefficient for commercial

scale.

Limitations of BEVS

Baculovirus Successes (National Library of Medicine (US), NCBI;2008)

• Alpha and beta interferon

• Adenosine deaminase

• Erythropoietin

• Interleukin 2

• Poliovirus proteins

• Tissue plamsinogen activator (TPA)

SSX2 tumor antigen (R.G. Kyyamova et. al. 2006)

Vaccines produced……. Blue Tongue disease

Porcine parvovirus

African equine fever

Avian Influenza

H1N1 Vaccine

Rota Virus Vaccine

Swine fever vaccine

Condt……. Name of vaccine Proteins Expressed Remarks

Swine Fever Vaccine Gp 55 Induce immunity and protection against virulent CSFV.

Influenza A/H1N1 Vaccine Heamagglutinin Alternatively Neuraminidase and Matrix

Equine influenza strain, A/equine/LP/93

Hemagglutinin (HA)

Rota Virus Like Particle VP2, VP6, and VP7 Sf9 cell, a host of the baculovirus.

Blue Tongue Vaccine NS2

Virus Like Particle

hemagglutinin (HA), neuraminidase (NA), and matrix (M1).

VLPs elicit antibodies that recognize a broader panel of antigenically distinct viral isolates compared to other vaccines in the HAI

Gene therapy It is applied in human medicine to a greater extent.

viz., Treatment of diabetes, hepatitis etc.

However, in veterinary sciences it is yet……..

Blue Tongue Diagnosis

Antigen Capture Competitive Enzyme-Linked Immunosorbent Assays Using Baculovirus-Expressed Antigens for Diagnosis of Bluetongue Virus and Epizootic Hemorrhagic Disease Virus .

(J. O. Mecham et al. 2003)

The genes coding for VP7 of BTV-11 and EHDV2 are reverse transcribed

FMD diagnosis The baculovirus expressed 2C acts as a suitable antigen

for the development of a reliable diagnostic test.

The sera of convalescent animals contain antibodies to 2C, a highly conserved non-structural protein, whereas the sera of vaccinated animals do not.

Nipah virus glycoprotein production (M. Eshaghi, et al.2004)

DNA encoding truncated G protein of NiV is cloned into the pFastBac HT vector, and the fusion protein to His-tag is expressed in insect cells by recombinant baculovirus.

The resulting His-G recombinant fusion protein is purified by affinity chromatography and used as the coating antigen for serological testing by indirect enzyme-linked immunosorbant assay (ELISA).

Economized large-scale production of high yield of rAAV

Large-scale production of rAAV remains one of the major challenges for continued development of pre-clinical and clinical studies, and for its potential commercialization.

This technology uses three different BEVs (Bac-Rep, Bac-GFP, and Bac-VP).

Miscellaneous…..

The baculovirus-insect cell expression system is widely used to produce recombinant proteins, including glycoproteins, for various biomedical applications.

Serological diagnosis of equine influenza using the hemagglutinin protein produced in a baculovirus expression system (Takeo Sugiura et al. 2001)

Current Status of BEVS…

Recent advances in baculovirus expression vector technology include improvements to methods for the selection of recombinant viruses and further developments in virion display vectors.

Baculovirus vectors are continue to be modified to facilitate gene expression in mammalian cells.

Current Status…….

The broad recognition and acceptance by the scientific community is reflected in the determination by The Institute of Scientific Information (ISI) that Dr. Summers is one of the top 250 most highly cited microbiologists in the world.

Acceptance of the BEVS by the private sector is reflected by the commercial licenses worldwide that are held for the BEVS technology (currently >70).

Conclusions Baculovirus is one of the most important eukaryotic

expression system.

This system is capable of generating large quantities of biologically active recombinant protein inexpensively and quickly.

The efficiency, low cost and large-scale production of proteins using BEVS represents breakthrough technology that is facilitating high-throughput proteomic studies.

Condt……

To date, over a thousand proteins have been expressed using the BEVS, with 98% being biologically active.

Baculovirus has advantage to permit post translational modifications of the protein expressed

Recombinant baculovirus have become widely used as vectors to express heterologous genes in cultured insect cells and insects larvae.

This gene produces up to 60% of the total protein of the virus and can be replaced by foreign genes.

Condt…… Baculoviruses are noninfectious to vertebrates and

their promoter have been shown to be inactive in most mammalian cells.

This trait gives them a real advantage in molecular biology when the protein is destined for diagnostic, targeted gene therapy or in medicine

The BEVS has become a core technology for:

1) The cloning and expression of genes for study of protein

structure, processing and function;

2) The production of biochemical reagents;

3) The study of regulation of gene expression;

4) The commercial exploration, development and production of

vaccines, therapeutics and diagnostics;

5) Drug discovery research;

Condt…..

Thank You for attention…..

……….Standby for discussion !!!!!