Research Update:

Identifying and Characterizing First

Infected Cells in the Rectum
Danijela Mari
March 11, 2015

Schematic Representation of Rectal

Tissue

Nature reviews

Research Goals
To detect transduced cell foci in the
rectal tissue using In Vivo Imaging
System (IVIS)
To identify transduced cells in tissue
sections and validate them using
antibody staining and Spectral Imaging
To understand how viral envelopes
determine target cell tropism in the
rectal tissue

Research Design
Lich Generation 2 reporter virus containing Luciferase and iRFP670
reporters was used
Several biopsies in the rectal compartment were introduced at
random
Female Rhesus Macaques were rectally challenged with Lich virus
containing either JR-FL or JR-CSF envelope
Forty-eight hours post challenge animals were sacrificed, recta were
removed and shipped to NU on ice overnight
Tissue was imaged using IVIS before and after addition of luciferin
to establish the level of background luminescence in the tissue
Areas with persistent luciferase signal post luciferin were cut out
and preserved at -80 degrees C in OCT for subsequent sectioning,
staining and imaging
The infected cells are validated by antibody staining, spectral
imaging and nested PCR and will be subsequently phenotyped

Infection of TZM-bl Cells with pLi670

VSVg Virus
LT
R

iRFP720

CM
V

Lucifera
se

IRES

iRFP67
0

WPRE

LT
R

iRFP670

iRFP702
iRF670 bleed through in mCherry is minimal

Spectral Profile of
pLi670
Transduced Cell

Wes Grimm

Research Goals
To detect transduced cell foci in the
rectal tissue using In Vivo Imaging
System (IVIS)
To identify transduced cells in tissue
sections and validate them using
antibody staining and Spectral Imaging
To understand how viral envelopes
determine target cell tropism in the
rectal tissue

Tissue Analysis Using IVIS

Luciferin
RM HP63 (JR-FL)
PBS

HP63_
2

HP63_
5

HP63_
5C

HP63_
5E

HP63_
1

HP63_
4

HP63_
5D

HP63_
5F

HP63_
5A

HP63_
5B

HP63_ 6

HP63_
3

HP63_
6C

HP63_
Biopsy

HP63_
6A

HP63_
6B

Tissue Analysis Using IVIS

Luciferin
RM GG70 (JR-FL)
PBS
GG70_
1

GG70_
3

GG70_
6C

GG70_
2

GG70_
6

GG70_
Biopsy

GG70_
5

GG70_
6D

GG70_ 6B

GG70_
4

GG70_ 6A

GG70_ 5
reimaged

Tissue Analysis Using IVIS

Luciferin
RM HP63 (JR-CSF)
PBS
FN94_4

FN94_2

FN94_1

FN94_6

FN94_
Biopsy

FN94_5

FN94_
3

FN94_
2A

FN94_2
B

FN94_2
E

FN94_2
F

FN94_2
D

FN94_2
C

Research Goals
To detect transduced cell foci in the
rectal tissue using In Vivo Imaging
System (IVIS)
To identify transduced cells in tissue
sections and validate them using
antibody staining and Spectral Imaging
To understand how viral envelopes
determine target cell tropism in the
rectal tissue

RM HP6 Piece 6A

The infected cell is in

epithelia:
Flower like ring
structures of the rectal
tissue are pointed by
yellow arrows
IVIS:
HP63_
6A

RFP 670 (Direct

Fluorescence)

RM HP6 Piece 6A: Volume View Movie

RFP 670 (Direct

Fluorescence)

RM HP6 Piece 6A: Cell 1

RFP 670 (Direct

Fluorescence)
DAPI

Luciferase:FITC
DAPI

RM HP6 Piece 6A: Spectral Profile

iRFP 670 Spectral Profile:

Expected Maximum Emission:
670nm
Observed Maximum Emission:

FITC Spectral Profile:

Expected Maximum Emission:
519nm
Observed Maximum Emission:

RM HP6 Piece 6A:

More Examples of Infected Cells

RFP 670 (Direct Fluorescence)

DAPI

RFP 670 (Direct

Fluorescence)
DAPI

RFP 670 (Direct

Fluorescence)
Luciferase:FITC
DAPI

Luciferase:FITC
DAPI

Luciferase:FITC
DAPI

GG70 Rectal Biopsy: Cell 1

IVIS:
GG70_
Biopsy

iRFP670 (direct
fluorescence)
DAPI

Luciferase:TRI
TC
DAPI

iRFP670 (direct
fluorescence)
Luciferase:TRITC
DAPI

GG70 Rectal Biopsy: Spectral Profile

iRFP 670 Spectral Profile:

Expected Maximum Emission:
670nm
Observed Maximum Emission:

FITC Spectral Profile:

Expected Maximum Emission:
519nm
Observed Maximum Emission:

Research Goals
To detect transduced cell foci in the
rectal tissue using In Vivo Imaging
System (IVIS)
To identify transduced cells in tissue
sections and validate them using
antibody staining and Spectral Imaging
To understand how viral envelopes
determine target cell tropism in the
rectal tissue

Cell Phenotyping
Phenotyping transduced cells in the tissue by staining
for various cell surface markers (CD3, CD4, CD68,
Th17,etc)
Optimized tissue digestion protocol involving
collagenase treatment and mechanical digestion
Successfully isolated cells from rectal tissue (1x1 cm
piece of tissue yields ~20 million of cells, up to half
were CD4+ T cells)
Optimized freezing and storing protocol for the
primary cells (over 90% cell viability was achieved
post freezing and storing at -80 degrees C, 3 months
later)
Infection of rectal cells with mCherry Lich virus
resulted in ~1% cells expressing mCherry protein

Building the Better

Reporter Constructs..

Generation of Lich Gen3 HSA

LT
R

CM
V

Lucifera
se

IRES

HS mCherr paGF
A
y
P

WPRE

Use HSA tag to sort for the transduced

cells isolated from the tissue
Direct mCherry to the membrane so that
localization of Luciferase and mCherry
would be distinct

LT
R

293T Cells Transfected

with GenX DNA
Western Blot Analysis

75
kDa

293T Cells Transduced

with GenX VSVg Virus
Immunofluorescence
Analysis

HSA:mCherry:GFP
(70kDa)

50
kDa
37
kDa

HSA/mCherry
Luciferase
DAPI

25
kDa

Anti-HSA
WB

Expected Sizes:
HSA: 15 kDa
mCherry: 27 kDa
paGFP: 28 kDa

Expected Localization:
HSA and mCherry: Membrane
Luciferase: Cytosolic

Generation of Lich GenX

LT
R

CM
V

HS mCherr Lucifera
A
y
se

WPRE

LT
R

Use HSA tag to sort for the transduced

cells isolated from the tissue
Ensure that fluorescent protein and
Luciferase expression go hand in hand

293T Cells Transfected with GenX DNA

Western Blot Analysis
Anti-mCherry

75
kDa
50
kDa
37
kDa
25
kDa

Anti-HSA

75
kDa
mCherry/HSA 50
kDa
37
mCherry

Expected Sizes:
HSA: 15 kDa
mCherry: 27 kDa
Luciferase: 50 kDa

kDa
25
kDa

Anti-Luciferase

100
kDa
75
HSA/mCherry
kDa
50
kDa
37
kDa
25
kDa

Lucifearase/mCherry/
HSA
Lucifearase/HS
A

In Depth Sequencing of GenX Lich

confirmed that all components of
the vector including LTRs , CMV
promoter, Triple Fusion Protein,
WPRE are present and of correct

Comparing the proteins expression after

transduction with Gen2 and GenX Lich VSVg
virus in 293T cells
Gen2 Lich: LT
R

CM
V

Lucifera
se

IRES

mCherr WPRE
y

LT
R

GenX Lich: LT CM HS mCherr Lucifera WPRE LT

R
V
A
y
se
R
Use various amounts of Gen2 and GenX Lich
DNA to make virus (1-6g of DNA)
Transduce 293 T cells with Gen2 and GenX VSVg
virus
Monitor expression of Luciferase and mCherry
proteins under the same experimental conditions

Gen2 Lich (4g) Transduced 293Ts

mCherry (direct
fluorescence)
Luciferase:Zenon 647
DAPI

GenX Lich (4g) Transduced 293Ts

mCherry (direct
fluorescence)
HSA:FITC
HSA and mCherry were coDAPI
expressed in all cells examined
No Luciferase expression was
detected in these cells

GenX Lich (4g) Transduced

mCherry (direct
fluorescence)
293Ts
Luciferase:Zenon 647
DAPI

Generation of Lich GenY

LT CM Lucifera IRE HS iRFP67 WPRE LT
R
V
se
S
A
0
R
LT CM Lucifera IRE HS mCherr WPRE LT
V
se
S
A
y
R
RGenerate
vectors
expressing
other
fluorescence
proteins (e.g. mCherry instead of iRFP670)
Infect animals with both viruses and study:
how viral envelopes determine target cell tropism
(rectal transmission and other projects)
the role of VPX in target cells preference (other
projects)
perform time course experiments (other projects)

State of the Project

Rectal biopsies are crucial for identification of infected
cell foci by IVIS
IVIS can direct us to the regions within the tissue that
contains the infected cells
Infected cells in the rectum were confirmed by antibody
staining and spectral imaging; nested PCR is in works
Phenotyping of infected cells in the tissue is currently in
progress
We have optimized protocol for isolation, freezing down
and infection of primary cells isolated from rectal tissue
Luciferase gene cloned between CMV and IRES is optimal
for Luciferase expression while HSA in front of the
mCherry increases expression of mCherry and renders it
membranous
Cloning of mCherry and iRFP670 variants that have such