Download To obtain cell-targeting specificity, the surface protein DC

Lili Yang1,3, Haiguang Yang2,3, Kendra Rideout2, Taehoon Cho2, Kye il Joo2, Leslie Ziegler2, Abigail
Elliot1,Anthony Walls1, Dongzi Yu1, David Baltimore1 & Pin Wang. “Engineered lentivector targeting
of dendritic cells for in vivo immunization”. Nature Biotechnology V 26.3, March 2008
Jared Toupin
Biology 509
Dr. Mourad
10 November 2010
Introduction
As an antigen presenting cell, a dendritic cell plays the crucial role of stimulating the
antigen-specific T-cell to elicit an adaptive immune response. Researchers Yang et al.
sought to manipulate this relationship by using a lentiviral vector(FUGW) to genetically
modify dendritic cells into presenting specific antigens to host T cells to produce a strong
antigen-specific T-cell response as well as an antibody response. By adding an
engineered glycoprotein(SVGmu) derived from the Sindbis virus to a lentiviral vector,
the dentritic cell-specific surface protein DC-SIGN was targeted both in vitro and in
vivo to determine the specificity, efficiency, and effectiveness in genetically modifying
dendritic cells. Finally, ovalbumin(OVA)-expressing E.G7 tumors cells were selected to
determine if dendritic cell-targeted lentivectors, containing the gene for FOVA, were
capable of eliciting an immune response to prevent or correct tumor formation.
Expirement/Results
To obtain cell-targeting specificity, the surface protein DC-SIGN of dendritic cells was
selected as the target for lentivector adhesion and subsequent endocytosis. The lentiviral
vector FUGW was pseudotyped with the Sinbis virus envelope glycoprotein (SVG) that
was specific for DC-SIGN. FUGW also contained a GFP reporter gene which was
quantified using flow cytometry. However, SVG also contained a receptor for heparin
sulfate, a commonly expressed protein in cells. To prevent promiscuous binding
throughout the host, SVG was engineered to SVGmu to disable the heparin sulfate
receptor. Subsequent green fluorescent protein (GFP)-viral protein R (vpr) labeling
showed over 70% of the lentiviral particles displayed the desired SVGmu protein.
In vitro analysis of FUGW/SVGmu transduction
From the 293T cell line, two cell lines human (293T.hDCSIGN) and murine
(293T.mDSIGN) were constructed to express DC-SIGN. Wild-type SVG and modified
SVGmu were then transduced using the lentiviral vector FUGW and the efficiency was
measured using flow cytometry to analyze the previously incorporated GFP gene
expression. The wild-type SVG had similar transduction efficiency (11% -16%) in all
three cell lines. The FUGW/SVGmu had (34% - 42%) transduction efficiency in only the
DC-SIGN containing cells, while the control DC-SIGN- 293T cells had only a small level
of transduction. Next, using a mixed mouse bone marrow culture containing 10% of
murine CD11c+ DC-SIGN+ dendritic cells, FUGW/SVGmu was determined to transduce
rougly 2% of those cells. Of the 2% of transduced cells, 95% were DC-SIGN+ CD11c+
which further indicated a high specificity for the DC-SIGN receptor. Finally, flow
cytometry analysis of mouse bone marrow derived dendritic cells (mBMDCs) showed
elevated expression of dendritic cell activation markers of CD86 and I-Ab on GFP+ cells
in comparison to GFP- cells after treatment of FUGW/SVGmu.
In vivo analysis of FUGW/SVGmu transduction
A B6 mouse was injected subcutaneously in the right flank with FUGW/SVGmu. After 3
days, the right inguinal lymph node near the injection site was found to contain almost a
tenfold increase in cell number when compared to the opposite side lymph node. Flow
cytometry of the lymph node found that approximately 3.2% of the CD11c+ cells
contained the GFP+ expression. Additionally when a comparison was made between
SVGmu, SVG, and VSVG (additional control), it was found that the FUGW/SVGmu
injection created a significantly larger lymph nodes with more traduced GFP+ dendritic
cells than the others. Subsequent analysis of the in vivo transduction using luciferase for
bioluminescence imaging proved unsuccessful as it was believed that the small
distribution of the dendritic cells were outside the sensitivity of the imaging.
In vitro CD8+ and CD4+ T-cell activation as well as in vivo IgG antibody response
A new lentivector was constructed to contain the gene for the antigen FOVA which
functions as an activator for CD8+ and CD4+ T-cell response. Secretion of IFN-γ from
both CD8+ and CD4+ T-cell as measured by ELISA indicated an immune response from
the FOVA/SVGmu lentivector in the dentritic cell . On the contrary FUGW/SVGmu
failed to elicit the same response indicating the effective delivery of antigens by the
dendritic cell. Additionally, following FOVA/SVGmu transduction, FOVA specific IgG
levels were analyzed in vivo in mouse spleen cells using ELISA. The serum titer for IgG
dramatically increased from 1:10,000 on day 7 to 1:30,000 on day 14. On the other hand
when FOVA/VSVG or FOVA/SVG was used, both generated less of a serum titer for
IgG between the two time periods.
Measuring the effectiveness of dendritic cells in conferring anti-tumor immunity
The E.G7 tumor model was selected to determine the effectiveness of dendritic celltargeted lentivectors in inducing immunization against tumor formation. The previously
used antigen, FOVA, was also used as a tumor antigen for this model. B6 mice were
initially injected with FOVA/SVGmu subcutaneously. After two weeks E.G7 tumor cells
were introduced into the mouse. The FOVA/SVGmu mice were completely protected
from tumor formation, whereas the mice that received a vaccination lacking the FOVA
transgene exhibited rapid tumor formation. The experiment was reversed to see if
injecting already growing E.G7 tumor cells in vivo could initiate an immune response to
the tumor formation. E.G7 tumor cells were expressed with a firefly luciferase gene
which was used to monitor tumor activity using bioluminescence imaging. By day 18
after the injection with the FOVA/SVGmu lentivector the mice were completely disease
free with no observed tumor relapse.
Conclusion:
While the results of using dendritic cell-targeted lentivectors to induce an effective
immune response are promising, much work has to be done surrounding the area of
lentivectors for in vivo immunization. However, by using the engineered glycoprotein
SVGmu, Yang et al. successfully demonstrated a strong specificity to the DC-SIGN
surface protein of dendritic cells both in vitro and in vivo. Subsequent flow cytometry of
the GFP gene incorporated into the FUGW/SVGmu showed a strong correlation between
dendritic cells possessing DC-SIGN and the eventual incorporation of the of the desired
gene product (GFP). One interesting question is why exactly does the SVGmu
dramatically increase the transduction of the desired gene over the wild type as both
contain the same receptor protein for the DC-SIGN protein. Additionally, it was shown
that using lentivector psuedotyped for the FOVA, dramatically increase both CD8+ and
CD4+ T-cell proliferation as well as IFN-γ production. Additional research should
continue as to why there was such a dramatic and sustained increase in the serum titer for
IgG upon exposure of the FOVA/SVGmu lentivector. Perhaps the most promising
conclusion from this research is the use of the lentivector psuedotyped with a specific
tumor antigen to induce vaccination or treatment for tumor formation. However, as the
authors point out the tumor’s antigen they selected in highly immunogenic which could
correlate to the dramatic reduction in the tumor formation. Additional research should be
undertaken with an assortment of antigens to truly understand the full potential of this
fascinating method.