Research  Update:    Iden0fying  and  Characterizing  First  Infected  Cells  in  the  Rectum  

Danijela  Marić  March  11,  2015  

Schema0c  Representa0on  of  Rectal  Tissue  

Nature  reviews  

Research  Goals  

•  To  detect  transduced  cell  foci  in  the  rectal  0ssue  using  In  Vivo  Imaging  System  (IVIS)  

•  To  iden0fy  transduced  cells  in  0ssue  sec0ons  and  validate  them  using  an0body  staining  and  Spectral  Imaging  

•  To  understand  how  viral  envelopes  determine  target  cell  tropism  in  the  rectal  0ssue  

Research  Design  •  Lich  Genera0on  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  aWer  addi0on  of  luciferin  

to  establish  the  level  of  background  luminescence  in  the  0ssue  •  Areas  with  persistent  luciferase  signal  post  luciferin  were  cut  out  

and  preserved  at  -­‐80  degrees  C  in  OCT  for  subsequent  sec0oning,  staining  and  imaging  

•  The  infected  cells  are  validated  by  an0body  staining,  spectral  imaging  and  nested  PCR  and  will  be  subsequently  phenotyped  

Infec0on  of  TZM-­‐bl  Cells  with  pLi670  VSVg  Virus  

iRF670  bleed  through  in  mCherry  is  minimal  

iRFP670

iRFP702

iRFP720

iRFP670 iRFP702Mock iRFP720

Relative brightness of iRFPs detected in Cy5 channel, Jake.

10x

40x

Ex/Em

Spectral  Profile  of  pLi670    Transduced  Cell  

Wes  Grimm  

LTR   CMV   iRFP670   WPRE   LTR  IRES  Luciferase  

Research  Goals  

•  To  detect  transduced  cell  foci  in  the  rectal  0ssue  using  In  Vivo  Imaging  System  (IVIS)  

•  To  iden0fy  transduced  cells  in  0ssue  sec0ons  and  validate  them  using  an0body  staining  and  Spectral  Imaging  

•  To  understand  how  viral  envelopes  determine  target  cell  tropism  in  the  rectal  0ssue  

 

Tissue  Analysis  Using  IVIS  RM  HP63  (JR-­‐FL)  

PBS  HP63_  2   HP63_  1  

HP63_  5   HP63_  4  HP63_  3  

HP63_  6  

HP63_  Biopsy  

Luciferin  

HP63_  5C   HP63_  5D   HP63_  5F  

HP63_  5E   HP63_  5A   HP63_  5B  

HP63_  6A  

HP63_  6B  

HP63_  6C  

           

Tissue  Analysis  Using  IVIS  RM  GG70  (JR-­‐FL)  

GG70_  6C   GG70_  6D   GG70_  6A  

GG70_  6B   GG70_  5  reimaged  

PBS  

GG70_  1   GG70_  2  

GG70_  3   GG70_  6   GG70_  5  

GG70_  Biopsy  

GG70_  4  

Luciferin  

Tissue  Analysis  Using  IVIS  RM  HP63  (JR-­‐CSF)  

PBS  FN94_4   FN94_1  

FN94_2   FN94_6   FN94_  3  

FN94_5  

FN94_  Biopsy  

Luciferin  

FN94_  2A   FN94_2B   FN94_2E  

FN94_2F   FN94_2D   FN94_2C  

Research  Goals  

•  To  detect  transduced  cell  foci  in  the  rectal  0ssue  using  In  Vivo  Imaging  System  (IVIS)  

•  To  iden0fy  transduced  cells  in  0ssue  sec0ons  and  validate  them  using  an0body  staining  and  Spectral  Imaging  

•  To  understand  how  viral  envelopes  determine  target  cell  tropism  in  the  rectal  0ssue  

 

RM  HP6  Piece  6A  

RFP  670  (Direct  Fluorescence)  DAPI  

The  infected  cell  is  in  epithelia:  Flower  like  ring  structures  of  the  rectal  0ssue  are  pointed  by  yellow  arrows  

IVIS:  HP63_  6A  

RM  HP6  Piece  6A:  Volume  View  Movie  

RFP  670  (Direct  Fluorescence)  DAPI  

RM  HP6  Piece  6A:  Cell  1  

RFP  670  (Direct  Fluorescence)  DAPI                                                                                              

Luciferase:FITC  DAPI                                                    

iRFP  670  Spectral  Profile:  Expected  Maximum  Emission:  670nm  Observed  Maximum  Emission:  669nm          

FITC  Spectral  Profile:  Expected  Maximum  Emission:  519nm  Observed  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:FITC  DAPI                                                                                              

Luciferase:FITC  DAPI                                                                                              

RFP  670  (Direct  Fluorescence)  Luciferase:FITC  DAPI                                                                                              

GG70  Rectal  Biopsy:  Cell  1  

iRFP670  (direct  fluorescence)  DAPI  

iRFP670  (direct  fluorescence)  Luciferase:TRITC  DAPI  

Luciferase:TRITC  DAPI  

IVIS:  GG70_  Biopsy  

GG70  Rectal  Biopsy:  Spectral  Profile  

iRFP  670  Spectral  Profile:  Expected  Maximum  Emission:  670nm  Observed  Maximum  Emission:  668nm          

FITC  Spectral  Profile:  Expected  Maximum  Emission:  519nm  Observed  Maximum  Emission:  520nm          

Research  Goals  

•  To  detect  transduced  cell  foci  in  the  rectal  0ssue  using  In  Vivo  Imaging  System  (IVIS)  

•  To  iden0fy  transduced  cells  in  0ssue  sec0ons  and  validate  them  using  an0body  staining  and  Spectral  Imaging  

•  To  understand  how  viral  envelopes  determine  target  cell  tropism  in  the  rectal  0ssue  

 

Cell  Phenotyping    •  Phenotyping  transduced  cells  in  the  0ssue  by  staining  for  various  

cell  surface  markers  (CD3,  CD4,  CD68,  Th17,etc)  •  Op0mized  0ssue  diges0on  protocol  involving  collagenase  

treatment  and  mechanical  diges0on    •  Successfully  isolated  cells  from  rectal  0ssue  (1x1  cm  piece  of  0ssue  

yields  ~20  million  of  cells,  up  to  half  were  CD4+  T  cells)  •  Op0mized  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)  

•  Infec0on  of  rectal  cells  with  mCherry  Lich  virus  resulted  in  ~1%  cells  expressing  mCherry  protein  

•  Isolate  RNA  from  the  cells  and  perform  RNA  sequencing  to  establish  the  nature  of  the  infected  cells  (in  future)  

Building  the  Beier  Reporter  Constructs..  

Genera0on  of  Lich  Gen3  HSA  

•  Use  HSA  tag  to  sort  for  the  transduced  cells  isolated  from  the  0ssue  

•  Direct  mCherry  to  the  membrane  so  that  localiza0on  of  Luciferase  and  mCherry  would  be  dis0nct  

LTR   CMV   mCherry   WPRE   LTR  HSA   paGFP  Luciferase   IRES  

293T  Cells  Transfected  with  GenX  DNA  Western  Blot  Analysis  

An0-­‐HSA  WB  

25  kDa  

50  kDa  

75  kDa  

37  kDa  

HSA:mCherry:GFP  (70kDa)  

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

HSA/mCherry  Luciferase  

DAPI  

293T  Cells  Transduced  with  GenX  VSVg  Virus  Immunofluorescence  Analysis  

Expected  Localiza0on:  HSA  and  mCherry:  Membrane  Luciferase:  Cytosolic  

Genera0on  of  Lich  GenX  

LTR   CMV   mCherry   WPRE   LTR  HSA   Luciferase  

•  Use  HSA  tag  to  sort  for  the  transduced  cells  isolated  from  the  0ssue  

•  Ensure  that  fluorescent  protein  and  Luciferase  expression  go  hand  in  hand  

293T  Cells  Transfected  with  GenX  DNA  Western  Blot  Analysis  

An0-­‐mCherry   An0-­‐HSA   An0-­‐Luciferase  

25  kDa  

37  kDa  

50  kDa  

75  kDa  

25  kDa  

37  kDa  

50  kDa  

75  kDa  

25  kDa  

37  kDa  

50  kDa  

75  kDa  100  kDa  

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

mCherry  

mCherry/HSA   Lucifearase/HSA  

Lucifearase/mCherry/HSA  

HSA/mCherry  

•  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  sequence  

Comparing  the  proteins  expression  aWer  transduc0on    with  Gen2  and  GenX  Lich  VSVg  virus  in  293T  cells  

•  Use  various  amounts  of  Gen2  and  GenX  Lich  DNA  to  make  virus  (1-­‐6μg  of  DNA)  

•  Transduce  293  T  cells  with  Gen2  and  GenX  VSVg  virus  

•  Monitor  expression  of  Luciferase  and  mCherry  proteins  under  the  same  experimental  condi0ons  

LTR   CMV   mCherry   WPRE   LTR  HSA   Luciferase  

LTR   CMV   mCherry   WPRE   LTR  IRES  Luciferase  Gen2  Lich:  

GenX  Lich:  

Gen2  Lich  (4μg)  Transduced  293Ts  

mCherry  (direct  fluorescence)  Luciferase:Zenon  647  DAPI  

GenX  Lich  (4μg)  Transduced  293Ts  

mCherry  (direct  fluorescence)  HSA:FITC  DAPI  

•  HSA  and  mCherry  were  co-­‐expressed  in  all  cells  examined  

•  No  Luciferase  expression  was  detected  in  these  cells  

mCherry  (direct  fluorescence)  Luciferase:Zenon  647  DAPI  GenX  Lich  (4μg)  Transduced  293Ts  

Genera0on  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  0me  course  experiments  (other  projects)  

LTR   CMV   iRFP670   WPRE   LTR  HSA  Luciferase   IRES  

LTR   CMV   mCherry   WPRE   LTR  HSA  Luciferase   IRES  

State  of  the  Project  •  Rectal  biopsies  are  crucial  for  iden0fica0on  of  infected  cell  foci  by  IVIS  •  IVIS  can  direct  us  to  the  regions  within  the  0ssue  that  contains  the  

infected  cells  •  Infected  cells  in  the  rectum  were  confirmed  by  an0body  staining  and  

spectral  imaging;  nested  PCR  is  in  works  •  Phenotyping  of  infected  cells  in  the  0ssue  is  currently  in  progress  •  We  have  op0mized  protocol  for  isola0on,  freezing  down  and  infec0on  

of  primary  cells  isolated  from  rectal  0ssue  •  Luciferase  gene  cloned  between  CMV  and  IRES  is  op0mal  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  arrangement  of  Luciferase  and  fluorescent  protein  is  underway