Title: Co-delivery of antigen and IL-12 by Venezuelan equine encephalitis virus replicon vectors enhances
antigen-specific immune responses and anti-tumor effects.
Journal: Cancer Immunology, Immunotherapy
Authors: Takuya Osada1, Peter Berglund5, Michael A. Morse2,4, Bolyn Hubby5, Whitney Lewis6, Donna
Niedzwiecki4, Xiao Yi Yang1, Amy Hobeika1, Bruce Burnett4, Gayathri R. Devi1,4, Timothy M. Clay1,3,4,, Jonathan
Smith5 , and H. Kim Lyerly1,3,4.
Affiliation: Duke University Medical Center, Departments of 1Surgery, 2Medicine, 3Immunology, and 4Duke
Comprehensive Cancer Center, Durham, NC 27710, U.S.A. 5Liquidia Technologies, RTP, NC 27709, 6Precision
Biosciences, Durham, NC 27701, U.S.A
Corresponding Author: H. Kim Lyerly, MDDuke University Medical CenterDepartment of SurgeryBox 2606 MSRB1 Rm 433b Research DrDurham, NC [email protected]: 919-684-5613
Supplementary Materials
Immunofluorescent staining of VRP-CEA(6D) infected Vero cells
Vero cells, grown to near confluency, were infected by removing the culture medium and overlaying the cell
monolayer with medium containing VRP-CEA(6D) or control HIV-gag VRP (moi 100), incubated overnight.
Productive infection was determined by staining for CEA expression under permeabilized and non-permeabilized
conditions followed by visualization by immunofluorescence. After fixation of cells with 4% paraformaldehyde for
10 min, permeabilization was performed with 0.01% triton x-100 in PBS for 7 min at room temperature.
Cells were labeled with mouse anti-CEA monoclonal antibody (Invitrogen, Carlsbad, CA) for 60 min, then with
FITC-conjugated anti-mouse IgG secondary antibody for 45 min. Similarly, IL-12 p70 expression by VRP-IL-12
infected Vero cells was verified using immunofluorescence staining (data not shown).
Western blot analysis of cell lysate from VRP-infected Vero cells.
Cell lysates were made from VRP-CEA(6D)-infected Vero cells, To confirm expression of full length CEA and
normal glycosylation of the molecule, a half of the cell lysate was incubated with/without Peptide N Glycosidase
F (PNGase F, 500 units/µg protein, Sigma) for 3 h at 37°C to deglycosylate CEA.
Similarly, Vero cells were infected with VRP-IL-12 (moi 100), incubated for 16 h, and cell lysate was made and
processed for SDS-PAGE. Western blot analysis was performed using an anti-mouse IL-12 antibody (Invitrogen).
Recombinant murine IL-12 (rIL-12) was loaded at various amounts (40, 80, 100, 200, 400 ng protein/lane) to
compare the separation of the p40 and p35 subunits in the denaturing gel with VRP-expressed monomeric
fusion polypeptide.
pERK3/CEA13200 bp
nsP1
nsP2
nsP4
KN(R)
nsP3CEA (6D)
26S P
T7 PCOLE1 ORI
Asc INot I
Eco RV
CEA replicon GAG replicon
non-permeabilized
permeabilized
A
C D
BA B
Lane: 1 2 3 4 CEA repliconRNA: + - + - PNGase: + + - -
C
Supplementary Figure 1
Supplementary Figure 1. CEA expression by VRP-CEA(6D) infected cells.
(A) VRP-CEA vector construct. (B) Vero cells, grown to near confluency, were infected by removing the
culture medium and overlaying the cell monolayer with medium containing VRP-CEA(6D) or control HIV-gag
VRP, incubated overnight. Productive infection was determined by staining for CEA expression under
permeabilized and non-permeabilized conditions followed by visualization by immunofluorescence (IFA). As
demonstrated, CEA staining was localized intracellularly as well as on the plasma membrane in cells
infected with VRP-CEA(6D). The non-cytoplasmic, reticular pattern of the intracellular signal was consistent
with biosynthesis through ER-Golgi for secretion on transmembrane insertion. (C) Western blot was used to
confirm the expression of full length CEA in lysates from VRP-CEA(6D) infected Vero cells. We confirmed
that full length CEA protein was expressed from the CEA replicon and yielded a shorter fragment consistent
with the putative 76.8 kDa molecular weight when deglycosylated. These data demonstrate that CEA is
appropriately translocated and post-translationally modified, and transported through the Golgi to the
plasma membrane.
pERK3/mIL-12(40-35)12682 bp
nsP1
nsP2
nsP4
KN(R)
nsP3
mIL12
26S P
T7 P
COLE1 ORI
Asc INot I
Eco RV
A B
Supplementary Figure 2
Supplementary Figure 2. IL-12 production by VRP-IL-12 infected cells.
(A) VRP-IL12 vector construct. The vector was designed to express murine IL-12 in the form of a single
polypeptide where the p35 and p40 subunits are fused into one open reading frame separated by a short linker
sequence derived from the elastin gene. (B) Functional p70 expression was verified using Western blot on cell
lysates from Vero cells infected with VRP-IL12. At 16 h post infection, cells were lysed and processed for SDS-
PAGE, and Western blot analysis using an IL-12 specific antibody. Cell lysate (pERK-IL12) was loaded in the
right-most lane and recombinant murine IL-12 (rIL-12) was loaded at various amounts (40, 80, 100, 200, 400
ng/lane), as indicated. rIL-12 migrates as two bands, corresponding to the separation of the p40 and p35
subunits in this denaturing gel, whereas the VRP-expressed monomeric fusion polypeptide appears as a 70
kDa band. Expected migration patterns of the polypeptides are indicated by the arrows.
Supplementary Figure 3
Mock
VRP-IL12(moi 100)
VRP-Empty
LPS
CD80 CD8673.2 79.8
87.8 453.6
87.1 553.7
76.3 185.7
Supplementary Figure 3. VRP-IL-12 infection of DC leads to maturation of DC.
Maturation status of VRP-IL-12 infected DC (MOI 10, 100) was analyzed after 24 hours of infection. Cells were
stained with FITC-labeled anti-mouse CD14, APC-labeled anti-I-A/I-E, and PE-lebeled anti-CD80 or anti-CD86
mAb. Blocking of Fcg receptor with anti-mouse CD16/CD32 was made before staining. CD14-negative/MHC
class II-positive DCs were analyzed for CD80/CD86 expression. Solid histograms show staining with PE-
labeled anti-CD80 or anti-CD86 mAb, and open histograms show control staining with PE-labeled control IgG.
Value of Mean Fluorescence Intensity is shown in each histogram.
CEA Antibody Response
CEA Rec
Prot (100
ng)
CEA RecPro
t+ IL-12
VRP (5e5
IU)10
100
1000
10000R
ecip
roca
l Tite
r (G
MT)
CEA Cellular Response
CEA Rec Protei
n (100
ng)
CEA RecPro
t+ IL-12
VRP (5e5
IU)
0
250
500
750
SFU
per
milli
on c
ells
CEA protein
CEA protein + VRP-IL
12
SFC
/1e6
lym
phoc
ytes
CEA protein
CEA protein + VRP-IL
12
Rec
ipro
cal T
iter
CEA Antibody Response CEA Cellular Response* *
Supplementary Figure 4
Supplementary Figure 4. Co-administration of VRP-IL-12 enhanced cellular and humoral immune
response against CEA with CEA protein vaccination.
To assess the immunomodulatory effect of VRP-IL-12 for a different vaccine platform, mice were
immunized with carcinoembryonic antigen (CEA(6D)) protein with/without co-injection of VRP-IL-12
(5x105 I.U). Immunization was repeated twice, 3 weeks apart. Immune monitoring assays were
performed 7 days after the second immunization. Anti-CEA immune responses were analyzed by IFN-
gamma ELISPOT assay or anti-CEA ELISA. VRP-IL-12 significantly enhanced anti-CEA cellular and
humoral immune responses induced by CEA(6D) protein vaccine. *p<0.01