RM HP63 Rectal Biopsy: Volume View Movie

Research Update: Identifying and Characterizing First Infected Cells in the RectumDanijela MariMarch 11, 2015

Schematic Representation of Rectal TissueNature reviews

The rectal mucosa is made up of a simple columnar epithelium that overlays a lamina propria that is dense in immune cells2

Research GoalsTo 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 ImagingTo understand how viral envelopes determine target cell tropism in the rectal tissue

Research DesignLich Generation 2 reporter virus containing Luciferase and iRFP670 reporters was used Several biopsies in the rectal compartment were introduced at randomFemale Rhesus Macaques were rectally challenged with Lich virus containing either JR-FL or JR-CSF envelopeForty-eight hours post challenge animals were sacrificed, recta were removed and shipped to NU on ice overnightTissue was imaged using IVIS before and after addition of luciferin to establish the level of background luminescence in the tissueAreas with persistent luciferase signal post luciferin were cut out and preserved at -80 degrees C in OCT for subsequent sectioning, staining and imagingThe 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

iRF670 bleed through in mCherry is minimal

Spectral Profile of pLi670 Transduced CellWes GrimmLTRCMViRFP670WPRELTRIRESLuciferase

Research GoalsTo 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 ImagingTo understand how viral envelopes determine target cell tropism in the rectal tissue

Tissue Analysis Using IVISRM HP63 (JR-FL)PBSHP63_ 2HP63_ 1HP63_ 5HP63_ 4HP63_ 3HP63_ 6HP63_ BiopsyLuciferin

HP63_ 5CHP63_ 5DHP63_ 5FHP63_ 5EHP63_ 5AHP63_ 5B

HP63_ 6AHP63_ 6BHP63_ 6C

Tissue Analysis Using IVISRM GG70 (JR-FL)

GG70_ 6CGG70_ 6DGG70_ 6AGG70_ 6BGG70_ 5 reimagedPBS

GG70_ 1GG70_ 2GG70_ 3GG70_ 6GG70_ 5GG70_ BiopsyGG70_ 4Luciferin

Tissue Analysis Using IVISRM HP63 (JR-CSF)PBSFN94_4FN94_1FN94_2FN94_6FN94_ 3FN94_5FN94_ BiopsyLuciferinFN94_ 2AFN94_2BFN94_2EFN94_2FFN94_2DFN94_2C

Research GoalsTo 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 ImagingTo understand how viral envelopes determine target cell tropism in the rectal tissue

RM HP6 Piece 6ARFP 670 (Direct Fluorescence)DAPIThe infected cell is in epithelia:Flower like ring structures of the rectal tissue are pointed by yellow arrows

IVIS:HP63_ 6A

RM HP6 Piece 6A: Volume View MovieRFP 670 (Direct Fluorescence)DAPI

RM HP6 Piece 6A: Cell 1

RFP 670 (Direct Fluorescence)DAPI Luciferase:FITCDAPI

iRFP 670 Spectral Profile:Expected Maximum Emission: 670nmObserved Maximum Emission: 669nm FITC Spectral Profile:Expected Maximum Emission: 519nmObserved Maximum Emission: 519nm RM HP6 Piece 6A: Spectral Profile

RM HP6 Piece 6A: More Examples of Infected Cells

RFP 670 (Direct Fluorescence)DAPI RFP 670 (Direct Fluorescence)DAPI Luciferase:FITCDAPI Luciferase:FITCDAPI RFP 670 (Direct Fluorescence)Luciferase:FITCDAPI

GG70 Rectal Biopsy: Cell 1

iRFP670 (direct fluorescence)DAPIiRFP670 (direct fluorescence)Luciferase:TRITCDAPILuciferase:TRITCDAPI

IVIS:GG70_ Biopsy

GG70 Rectal Biopsy: Spectral Profile

iRFP 670 Spectral Profile:Expected Maximum Emission: 670nmObserved Maximum Emission: 668nm FITC Spectral Profile:Expected Maximum Emission: 519nmObserved Maximum Emission: 520nm

Research GoalsTo 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 ImagingTo 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 proteinIsolate RNA from the cells and perform RNA sequencing to establish the nature of the infected cells (in future)

Building the Better Reporter Constructs..

Generation of Lich Gen3 HSAUse HSA tag to sort for the transduced cells isolated from the tissueDirect mCherry to the membrane so that localization of Luciferase and mCherry would be distinctLTRCMVmCherryWPRELTRHSApaGFP

LuciferaseIRES

293T Cells Transfected with GenX DNAWestern Blot Analysis

Anti-HSA WB25 kDa50 kDa75 kDa37 kDaHSA:mCherry:GFP (70kDa)Expected Sizes:HSA: 15 kDamCherry: 27 kDapaGFP: 28 kDa

HSA/mCherryLuciferaseDAPI293T Cells Transduced with GenX VSVg VirusImmunofluorescence AnalysisExpected Localization:HSA and mCherry: MembraneLuciferase: Cytosolic

Generation of Lich GenXLTRCMVmCherryWPRELTRHSALuciferase

Use HSA tag to sort for the transduced cells isolated from the tissueEnsure that fluorescent protein and Luciferase expression go hand in hand

293T Cells Transfected with GenX DNAWestern Blot Analysis

Anti-mCherryAnti-HSAAnti-Luciferase25 kDa37 kDa50 kDa75 kDa25 kDa37 kDa50 kDa75 kDa25 kDa37 kDa50 kDa75 kDa100 kDaExpected Sizes:HSA: 15 kDamCherry: 27 kDaLuciferase: 50 kDamCherrymCherry/HSALucifearase/HSALucifearase/mCherry/HSAHSA/mCherryIn 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 sequence

Comparing the proteins expression after transduction with Gen2 and GenX Lich VSVg virus in 293T cells

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 virusMonitor expression of Luciferase and mCherry proteins under the same experimental conditionsLTRCMVmCherryWPRELTRHSALuciferase

LTRCMVmCherryWPRELTRIRESLuciferaseGen2 Lich:GenX Lich:

Gen2 Lich (4g) Transduced 293TsmCherry (direct fluorescence)Luciferase:Zenon 647DAPI

GenX Lich (4g) Transduced 293TsmCherry (direct fluorescence)HSA:FITCDAPIHSA and mCherry were co-expressed in all cells examinedNo Luciferase expression was detected in these cells

mCherry (direct fluorescence)Luciferase:Zenon 647DAPIGenX Lich (4g) Transduced 293Ts

Generation of Lich GenY

Generate 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)LTRCMViRFP670WPRELTRHSALuciferase

IRESLTRCMVmCherryWPRELTRHSALuciferase

IRES

State of the ProjectRectal biopsies are crucial for identification of infected cell foci by IVISIVIS can direct us to the regions within the tissue that contains the infected cellsInfected cells in the rectum were confirmed by antibody staining and spectral imaging; nested PCR is in worksPhenotyping of infected cells in the tissue is currently in progressWe have optimized protocol for isolation, freezing down and infection of primary cells isolated from rectal tissueLuciferase 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 membranousCloning of mCherry and iRFP670 variants that have such arrangement of Luciferase and fluorescent protein is underway