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 27710Lyerl001@mc.duke.eduTel: 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