Interactions between ovine lentiviral vectors and primary cells · 2016-07-20 · Background...

Interactions between ovine lentiviral vectors and primary cells

Rebecca McLean Moredun Research Institute

MRP Inter-Institutional Post-Graduate Competition 28th/29th June 2016

www.moredun.org.uk

https://www.google.co.uk/url?q=http://boxing.eusu.ed.ac.uk/&sa=U&ei=RhUjU-mWNoeU0AXV24HYBg&ved=0CC0Q9QEwAA&usg=AFQjCNHimv0C2Aft15Iso8oSADk2M0l2GA

• Many infectious diseases of livestock do not yet have an effective vaccine

• Viral vectors

• Lentiviral vaccine vectors

– Ovine lentivirus

Livestock vaccines

Increasing safety profile Primary Cells Interactions with APCs Background Conclusions

BFV PFV

SFV-3

WEHV-1

WEHV-2

WDSV

GALV MMLV

FeLV RSV

FIV

BLV

ENTV-2

HTLV-1

HTLV-2

HIV-1

SIVmac

EIAV

VMV

MPMV

MMTV HERV-K

spumaretrovirus

deltaretrovirus lentivirus

betaretrovirus gammaretrovirus

epsilonretrovirus

alpharetrovirus

ENTV-1 JSRV

BIV

Retroviruses

Ovine lentivirus


Lentivirus Structure

Matrix Capsid Nucleo- capsid

Gag

Protease

Pro Surface Unit

Trans Membrane

Env

Reverse Transcriptase

RnaseH Integrase

Pol

R U5 U3 R U5 U3

Ψ

LTR LTR

Env SU

TM

Gag

CA

MA

NC

Pol IN

PR

RT

Pro

Vif Tat

Rev


Why lentiviruses?

• Can infect non-dividing antigen presenting cells (APCs)

• Long lasting, stable transgene expression

• Produce cellular immune responses1

• Infectious disease

• Generate neutralising antibodies2

1) Rowe et al. 2006. Mol Ther. 13. 2) Iglesias et al. 2006. J Gene Med. 8.


Lentivirus Replication Cycle • Easily manipulate genome

How will it work?

Nucleus

Cytoplasm

Lentiviral vaccine vector • Self-inactivating • Integration deficient

Antigen presentation


Aims


1. Construct and evaluate modified ovine lentiviral vaccine vectors

2. Study the innate immune response to ovine lentiviral vectors in primary ruminant dendritic cells and

macrophages cultured in vitro

Basic characterisation

Enhance safety profile

Improve production efficiency

Efficiency of infection

Immune responses

Interactions with primary cells

Vector Production

Harvest lentiviral vector

48 hours later...

Transfect human fetal kidney cell line

Infect target cells (CRFK)


Self-Inactivating


Gag

Protease

Pro Surface Unit

Trans Membrane


RnaseH Integrase

Pol

R U5 U3 R U5 U3

Ψ

3’ LTR 5’ LTR

Recombination events

LTR LTR eGFP

LTR LTR eGFP Promoter

LTR LTR eGFP

11.32%

13.65%

0.28%

eGFP SSC


Integration Deficient


Gag

Protease

Pro Surface Unit

Trans Membrane

Env


RnaseH Integrase

Pol

R U5 U3 R U5 U3

Ψ

LTR LTR

5751 GAGGGGAATA GATCATTGGC AAGTGGccTA CACTCATTTT GAAGATAAGA R G I D H W Q V D Y T H F E D K I 5801 TATTACTAGT ATGGGTAGAA ACAAATTCGG GATTAATTTA TGCAGAAAGG L L V W V E T N S G L I Y A E R 5851 GTGAAAGGGG AGACAGGACA AGAATTTAGA GTAACAGCTA TGAAGTGGTA V K G E T G Q E F R V T A M K W Y 5901 TGCTCTGTTT GCCCCAAAAT CATTGCAATC TGATAATGGG CCAGCATTTG A L F A P K S L Q S D N G P A F V 5951 TAGCAGAAGC AACACAACTG CTAATGAAAT ATTTAGGGAT AATACATACA A E A T Q L L M K Y L G I I H T 6001 ACAGGGATAC CTTGGAATCC ACAGTCTCAA GCTCTAGTGG ccAGGGCTCA T G I P W N P Q S Q A L V E R A H

D,D-35-E motif


Non-integrated DNA outcome

1-LTR episome 2-LTR episome

Provirus

Pre-Integration Complex

• Intact viral coding region

• Transgene expression

• Lack origin of replication (ORI)

U3 U5 R

U3 U5 R U3 U5 R

U3 U5 R U3 U5 R


Transgene

Transgene

Transgene

∆IN Vector - Results

• Dividing and non-dividing CRFK • Arrested using aphidicolin

• High multiplicity of infection (MOI=1)

• eGFP-positive cells measured every 2 days for 12 days 48 hours – 50% of cells released from cell cycle arrest


0

10

20

30

40

50

60

70

80

90

100

2 4 6 8 10 12

eG

FP P

osi

tive

Ce

lls

(% o

f d

ay 2

)

Time Post-Infection (Hours)

Parent Vector (Dividing Cells)

Parent Vector (Non-Dividing Cells)

ΔIN (Dividing Cells)

ΔIN (Non-Dividing Cells)

Improved Safety

• Self-inactivating (SIN)

• Integration deficient (∆IN)

• Combined and used to infect CRFK

• Flow analysis at 72 hours post infection


1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

Parent SIN+ΔIN

Vir

al T

itre

(IU

/ml)

Vector Construct

Virus Construct

Type Viral Titre

(IU/ml)

Reduction in titre compared

to Parent

HIV-1 Parent 6.2x109

270-fold1 SIN+ΔIN 2.3x107

EIAV Parent 9.3x108

290-fold1 SIN+ΔIN 3.2x106

VMV Parent 2.6x106

5-fold SIN+ΔIN 5.7x105

1) Ellis et al. 2012. Molecular Therapy–Nucleic Acids . 1.

107

106

105

104

103

102

101

100

Primary Cells

• Ovine peripheral blood

• CD14+ → IL-4 & GM-CSF

• Monocyte derived dendritic cells (DCs)

• Day 6: infect


Day 6 Day 9 Day 0 10x 20x 20x

HIV and VMV Vector comparisons

• HIV-1 derived lentiviral vectors commonly used

• MOI=1

• Analysed level of eGFP positive cells over time


0

10

20

30

40

50

60

70

80

90

100

1 2 3 4 6 8 10 12

eG

FP P

osi

tive

Ce

lls (

%)

Time Point (hours)

VMV Vector HIV Vector SIN-∆IN VMV Vector

High level of cell death

• Flow cytometry analysis

– 72 hours

– Only lifted off cells expressing eGFP

– Large percentage (>90%) of the cells expressing eGFP were dead.

Infected Cells 72 hours post-infection

• Mechanism of cell death

• Initial time points – 12 hours and 24 hours


56.50% 0.68%

14.51%

Annexin V

PI

Necrotic Late Apoptotic

Early Apoptotic

12 hours post-infection

Apoptosis

• Time course

• Naturally low levels of apoptosis in all controls

• Infected cells undergo apoptosis due to infection after 4 hours.

0

5

10

15

20

25

30

35

40

45

50

1 2 3 4 6 8 10 12

Earl

y A

po

pto

tic

Ce

lls (

%)

Time Point (hours)

Infected Heat Inactivated Control Uninfected Untreated


What’s inducing cell death?

DNA?

Vector which is unable to reverse transcribe the RNA does not induce apoptosis

YMDD AAAA

Sensing of DNA induces apoptosis

05

1015202530354045

1 2 3 4 6 8 10 12

Earl

y A

po

pto

tic

Ce

lls (

%)

Time Point (hours) Infected Untreated ∆RT Vector


Next Stages

• Some adjuvants induce apoptosis1

• Phagocytosis

• Cross presentation

Increasing safety profile Primary Cells Background

Primary cell population Apoptotic bodies

Secondary cell population

Cross presentation

1) Kool et al., 2008. J Immunology. 181.

Interactions with APCs Conclusions

Next Stages

Increasing safety profile Primary Cells Background

32.67%

18.67%

47.81%

PKH-67 (green)

PK

H-2

6

(red

)

Interactions with APCs Conclusions

+6 hours

+4 hours

Outputs


• Cloned in a range of pathogen genes:

• Chlamydia abortus

• Teladorsagia circumcincta

• Louping Ill Virus

• Toxoplasma gondii

• Improve vaccine efficiency

Conclusions

• Ovine lentiviral vectors are promising candidates for gene delivery in ruminants.

• Integrase deficient and self-inactivating.

• Primary cells can efficiently be infected in vitro.

• Infected cells undergo apoptosis after 4 hours of infection.

• Similar to action of some adjuvants.

• Apoptotic bodies can be phagocytosed.


Acknowledgements

Funders

Moredun Scientific Scottish Government

Moredun Research Institute

David Griffiths Gary Entrican

Ann Wood Sean Wattegedera

Mara Rocchi Maira Connelly Tom McNeilly

David Longbottom Helen Todd

BioServices David Kennedy

Roy Davie Manus Graham

The Roslin Institute

Jayne Hope Hope Lab Group

https://www.google.co.uk/url?q=http://boxing.eusu.ed.ac.uk/&sa=U&ei=RhUjU-mWNoeU0AXV24HYBg&ved=0CC0Q9QEwAA&usg=AFQjCNHimv0C2Aft15Iso8oSADk2M0l2GA

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Interactions between ovine lentiviral vectors and primary cells · 2016-07-20 · Background...

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