DNA vaccine encoding CD40 targeted to dendritic

cells in situ prevents the development of Heymann

nephritis in rats
Ya Wang
1,7
, Yuan Min Wang
2,7
, Yiping Wang
1
, Guoping Zheng
1
, Geoff Yu Zhang
2
,
Jimmy Jianheng Zhou
2
, Thian Kui Tan
1
, Qi Cao
1
, Min Hu
2
, Debbie Watson
2,3
, Huiling Wu
4
, Dong Zheng
1
,
ChangQi Wang
1
, Mireille H. Lahoud
5
, Irina Caminschi
5,6
, David C. Harris
1
and Stephen I. Alexander
2
1
Centre for Transplantation and Renal Research, University of Sydney at Westmead Millennium Institute, Westmead, Sydney, New South
Wales, Australia;
2
Centre for Kidney Research, Childrens Hospital at Westmead, Westmead, Sydney, New South Wales, Australia;
3
Centre for Medical Bioscience, University of Wollongong, Wollongong, New South Wales, Australia;
4
Collaborative Transplant Research
Group, Royal Prince Alfred Hospital, Camperdown, New South Wales, Australia;
5
Centre for Immunology, Burnet Institute, Melbourne,
Victoria, Australia and
6
Department of Microbiology and Immunology, University of Melbourne, Melbourne, Victoria, Australia
The CD40-CD154 costimulatory pathway has been shown to
be critical for both T- and B-cell activation in autoimmune
disease. Here, we assessed the effects of blocking this
pathway using CD40 DNA vaccine enhanced by dendritic cell
targeting on the development of active Heymann nephritis,
a rat model of human membranous nephropathy. DNA
vaccination delivers plasmid DNA encoding the target
antigen, either alone or in combination with enhancing
elements, to induce both humoral and cellular immune
responses. To determine whether CD40 DNA vaccine
targeting the encoded CD40 directly to dendritic cells would
improve the efficacy of the vaccination against self-protein
CD40, we utilized a plasmid encoding a single-chain Fv
antibody specific for the dendritic cellrestricted antigen-
uptake receptor DEC205 (scDEC), the target gene CD40, and
the adjuvant tetanus sequence p30. This vaccine plasmid
was compared to a control plasmid without scDEC. Rats
vaccinated with scDEC-CD40 had significantly less proteinuria
and renal injury than did rats receiving scControl-CD40 and
were protected from developing Heymann nephritis. Thus,
CD40 DNA vaccination targeted to dendritic cells limits the
development of Heymann nephritis.
Kidney International (2013) 83, 223232; doi:10.1038/ki.2012.374;
published online 5 December 2012
KEYWORDS: active Heymann nephritis (HN); CD40; DEC205; DNA vaccination
Membranous nephropathy, a major autoimmune cause of
chronic renal failure, is an immune disease driven in many
cases by the recently described cognate antigen PLA2R
expressed on the glomerular epithelium.
1,2
CD40 expressed
by B cells and other antigen-presenting cells and its ligand
CD154 on T cells are critical costimulatory pathways required
for T-cell activation and for B-cell differentiation and class
switching.
3
CD40-CD154 blockade has been shown to be protective in
a number of models of renal disease, including rodent
membranous glomerulonephritis and Adriamycin nephrop-
athy.
46
CD154 blocking antibodies were shown to be highly
effective in primate studies on transplantation and are used
clinically. However, their effect in activating CD154 on
platelets and subsequent thrombosis has limited their clinical
use.
79
CD40 antibodies can block this pathway without
associated thrombosis and are in preclinical testing, although
some are associated with depletion of CD40-expressing cells
including B cells.
1014
Recent studies suggest that blockade of
this pathway using antibodies generates antigen-specific Tregs
and limits antigen-specific CD8 expansion.
15
DNA vaccination delivers plasmid DNA encoding the
target antigen, either alone or in combination with enhancing
elements, to induce both humoral and cellular immune
responses. Characteristically, DNAvaccines have been used to
target foreign antigens; however, we have shown significant
benefits in different models of renal disease targeting self-
antigens including chemokines CCL2 (MCP-1) and specific
TCRs using this strategy, including the benefits of T
celldirected adjuvants in therapeutic plasmids.
16,17
The use
of DNA vaccination has been limited by poor immune
responses and this is a particular problem while generating
responses to self-antigens where there is intrinsic self-
tolerance.
18,19
A recent study by Steinman and others has
shown the benefits of targeting the DNA-encoded plasmids
to DCs using scFv antibodies directed at DEC205 on the DC
http://www.kidney-international.org bas i c r es ear ch
& 2012 International Society of Nephrology
Correspondence: Stephen I. Alexander, Centre for Kidney Research,
Childrens Hospital at Westmead, Westmead, Sydney, New South Wales
2145, Australia. E-mail: StephenA@chw.edu.au
7
These authors contributed equally to this work.
Received 4 May 2012; revised 30 July 2012; accepted 24 August 2012;
published online 5 December 2012
Kidney International (2013) 83, 223232 223
surface and we have used this approach in targeting CD40 to
DCs.
20
The ability to direct antigens to DCs has the potential
to enhance immunogenicity, which is crucial for DNA
vaccination and for generating responses against self-
antigens.
Active Heymann nephritis (HN), an experimental rat
model of human autoimmune-mediated membranous
nephritis, is induced in Lewis rats by immunization with a
crude renal tubular antigen (RTA/Fx1A) and reproduces
clinical features of human idiopathic membranous glomer-
ulonephritis.
21,22
Both T cell and B cellgenerated anti-
bodies have been shown to have a role in its etiology, and
thus CD40/CD154, a key costimulatory pathway between
T and B cells, is an attractive target for blockade by DNA
vaccination.
Here we assess the role of the CD40 DNAvaccine targeting
an encoded CD40 fused to a tetanus toxoid T-helper epitope
P30 to dendritic cells and evaluate its efficacy in protecting
against renal disease, the level of antibody produced, and its
functional characteristics in vivo and in vitro.
RESULTS
Construction and expression of scDEC-CD40 and
scControl-CD40 DNA vaccines
To generate DNA vaccine targeting the encoded CD40 to
DCs, the open reading frame for mouse CD40 extracellular
domain was cloned into pSC-DEC-OLLA vector (scDEC), a
modified pcDNA3.1 vector that contains a gene encoding
a single-chain antibody specific for mouse DEC205.
20
We
therefore generated a fusion DNA construct with the CD40
gene fused in frame to the C-terminus of scDEC as shown in
Figure 1a. Our previous studies showed that introduction of
the P30 epitope increased immunogenicity of CCL2 DNA
vaccination in a murine model of Adriamycin Nephrop-
athy.
17
We therefore included the P30 epitope at the
C-termimus of scDEC-CD40. As a control, CD40 DNA
vaccine that does not target the encoded CD40 to DCs
(scControl-CD40) was generated as described above using
pSC-GL117-OLLA (scControl) vector instead of scDEC
vector. The expression of the DNA constructs was
confirmed by immunoblotting of cell lysates from cells
transfected with respective plasmid DNA (Figure 1b). We
further demonstrated that scDEC-CD40 but not scControl-
CD40 fusion protein was able to bind to DEC205-expressing
CHO-K1 cells (Figure 1c), demonstrating the targeting of
scDEC-CD40 to DCs.
scDEC-CD40 DNA vaccination induces stronger anti-CD40
autoantibody response compared with scControl-CD40 DNA
vaccination
We measured the serum level of the anti-CD40 autoantibody
using the enzyme-linked immunosorbent assay (ELISA) to
assess the potency of the different vaccines. Rats were injected
intramuscularly (i.m.) twice with scDEC-CD40 or scControl-
CD40 DNAvaccines within a 3-week interval. One week after
the second vaccination, sera were collected and subjected to
ELISA analyses. Serum collected from nonvaccinated rats was
used as control. As shown in Figure 2a, there was a fourfold
increase in anti-CD40 autoantibody level in scDEC-CD40-
vaccinated rats as compared with scControl-CD40-vaccinated
scDEC-CD40
scControl-CD40
5
5
scDEC mCD40 (aa20-aa193) P30 3
scGL117 mCD40 (aa20-aa193) P30 3
50 kDa
1 3 2 4 5
1: Nontransfected HeLa cell lysate
2. scControl vector cell lysate
3. scDEC vector cell lysate
4. scControl-CD40 cell lysate
5. scDEC-CD40 cell lysate
CHO-DEC-205 transfectants CHO-parental cells
Unstained
N/T sup
Cont-CD40
DEC-205-CD40
0
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
PE
Figure 1| Design and expression of scDEC-CD40 and scControl-CD40 DNA vaccines. (a) Open reading frame for mouse CD40 extracellular
domain was fused in frame to the C-terminus of scDEC or scControl, and tetanus toxoid Thelper epitope P30 was fused in frame to the
C-terminus of CD40. (b) Plasmid DNAs (scControl-CD40, scDEC-CD40, scControl vector, and scDEC vector) were transiently transfected into
HeLa cells, and 24 h post transfection total cell lysates were subjected to western blot. (c) DEC205-expressing CHO-K1 cells (CHO-DEC-205
transfectants) or parental CHO-K1 cells (CHO-parental cells) were incubated with supernatant from HeLa cells either nontransfected (N/T) or
transfected with scControl-CD40 or scDEC-CD40 plasmid DNAs. Cells were washed after incubation and subjected to flow cytometry analysis
with PE rat anti-mouse CD40 antibody. A representative plot of two independent experiments is shown.
bas i c r es ear ch Y Wang et al.: DC-targeted CD40 DNA vaccine prevents HN
224 Kidney International (2013) 83, 223232
ones (0.52380.2216 vs. 0.11630.0724, Po0.01). The
serum level of the anti-CD40 autoantibody in scControl-
CD40-vaccinated rats was not significantly increased com-
pared with that in nonvaccinated ones.
We assessed the longevity of antibody production, the
elevation of anti-CD40, and measured anti-CD40 autoanti-
body levels at 4, 6, 8, 10, and 12 weeks after induction of HN.
Rats were divided into four groups as described in Materials
and Methods: DEC-CD40-HN, con-CD40-HN, HN, and
complete Freunds adjuvant (CFA). There was a persistence of
significantly elevated levels of CD40 antibody throughout the
time course of HN in the DEC-CD40-HN group (Figure 2b).
The anti-CD40 autoantibody levels in the con-CD40-HN
group and HN group were low and not significantly different
throughout the time course of HN.
CD40 DNA vaccination protects renal function
To assess the effects of CD40 vaccination on the development
of HN, we measured total urinary protein over 16 h, urine
creatinine, serum albumin, and creatinine at 6, 8, 10, 12
weeks post Fx1A/CFA immunization. The DEC-CD40-HN
group did not develop proteinuria throughout the 12-week
period. Proteinuria (urine protein/creatinine) of the DEC-
CD40-HN group was equivalent to that of the control CFA
group throughout the time course (Figure 3a). In the con-
CD40-HN group, the onset of proteinuria was delayed by
2 weeks as compared with the HN group, demonstrating an
effect, although less potent compared with the DEC-modified
vaccine. At week 12, the DEC-CD40-HN group had
significantly higher serum levels of albumin compared with
the con-CD40-HN and HN groups (Figure 3b) and the
serum creatinine level at week 12 was significantly lower in
the DEC-CD40-HN group than in the HN group (Figure 3c).
No significant differences were observed in terms of
creatinine clearance among all four groups at week 12 (data
not shown). Our results showed that both scControl-CD40
Anti-CD40 autoantibody
** ** 0.8
0.6
0.4
0.2
0.0
A
b
s
o
r
b
a
n
c
e
con-CD40
vaccine
DEC-CD40
vaccine
No vaccine
Anti-CD40 autoantibody
0.6
0.4
0.2
0.0
4 6 8
Weeks
10 12
A
b
s
o
r
b
a
n
c
e

(
n
m
)
con-CD40-HN
DEC-CD40-HN
HN
CFA
*
*
*
*
*
Figure 2 | Anti-CD40 autoantibody level in serum measured by
enzyme-linked immunosorbent assay (ELISA). (a) Sera were
collected from rats either vaccinated (scControl-CD40, n 5 or
scDEC-CD40, n 5) or nonvaccinated (n 4) 1 week after the second
vaccination and 1 week before Fx1A/complete Freunds adjuvant
(CFA) immunization. All the sera were diluted 10 and subjected to
ELISA. Absorbance was read at 450 nm corrected against background
reading at 570 nm. The scDEC-CD40 vaccine induces significantly
higher serum anti-CD40 autoantibody levels as compared with the
scControl-CD40 and nonvaccinated groups (**Po0.01). (b) Serum
anti-CD40 autoantibody was measured at weeks 4, 6, 8, 10, and 12
post Fx1A/CFA immunization for four groups: the con-CD40-HN
group (rats vaccinated with scControl-CD40 DNA, followed by Fx1A
immunization, n 5); the DEC-CD40-HN group (rats vaccinated
with scDEC-CD40 DNA, followed by Fx1A immunization, n 5); the
Heymann nephritis (HN) group (nonvaccinated rats immunized with
Fx1A, n 4); and the CFA group (nonvaccinated rats immunized with
CFA, n 3). Serum anti-CD40 autoantibody levels were significantly
higher in the DEC-CD40-HN group than in the other three groups at
all five time points (*Po0.05). All results are expressed as mean

s.d.
8000
6000
4000
2000
0
con-CD40-HN
DEC-CD40-HN
HN
CFA
6 8 10 12
U
r
i
n
e

p
r
o
t
e
i
n
/
c
r
e
a
t
i
n
i
n
e

(
g
/
m
o
l
)
**
#
**
*
#
**
Weeks after disease induction
40
30
20
10
0
** **
#
*
#
S
e
r
u
m

a
l
d
u
m
i
n

(
g
/
l
)
c
o
n
-
C
D
4
0
-
H
N
D
E
C
-
C
D
4
0
-
H
N
H
N
C
F
A
c
o
n
-
C
D
4
0
-
H
N
D
E
C
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D
4
0
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C
F
A
100
80
60
40
20
0 S
e
r
u
m

c
r
e
a
t
i
n
i
n
e

(

m
o
l
/
l
)
**
Figure 3| CD40 DNA vaccination protects against renal failure.
Urine and serum were collected at weeks 6, 8, 10, and 12 post Fx1A/
complete Freunds adjuvant (CFA) immunization and urine/serum
creatinine and serum albumin were measured. (a) Urine protein:
creatinine ratio: week 8, **Po0.01 between Heymann nephritis (HN)
and the other three groups; weeks 10 and 12,
#
Po0.001 between
HN and DEC-CD40-HN or CFA groups. (b) Serum albumin. (c) Serum
creatinine. All results are expressed as mean

s.d. (*Po0.05;
**Po0.01;
#
Po0.001).
Kidney International (2013) 83, 223232 225
Y Wang et al.: DC-targeted CD40 DNA vaccine prevents HN bas i c r es ear ch
vaccination and scDEC-CD40 vaccination protected HN rats
with a reduction in proteinuria; however, scDEC-CD40
vaccination was significantly more protective compared with
scControl-CD40 vaccination.
CD40 DNA vaccination reduces renal structural injury
Renal structural injury was assessed histologically by
evaluating glomerulosclerosis and tubular atrophy
(Figure 4a). Both con-CD40-HN and DEC-CD40-HN groups
had significantly less glomerulosclerosis and tubular atrophy
compared with the HN group (Figure 4b).
scDEC-CD40 DNA vaccination reduces immune cell
infiltration and IgG deposition
Immune cell infiltrations were assessed by immunohisto-
chemical staining of kidney sections with antibodies specific
for CD4, CD8, and macrophages at week 12 post Fx1A/CFA
immunization. As shown in Figure 5a, the DEC-CD40-HN
group had significantly less CD4

and macrophage cell

infiltrates compared with the HN group, whereas the con-
CD40-HN group was not significantly different compared
with the HN group. CD8

cell infiltration did not differ

significantly among the four groups. Figure 5b shows
representative histology from each group. Furthermore,
glomerular immunoglobulin G (IgG) deposition (one of
the major characteristics of HN) in the DEC-CD40-HN
group was less dense compared with the other HN groups
(Figure 5c). Instead of a continuous ribbon-like staining of
IgG, as seen in HN rats, rats in the DEC-CD40-HN group
displayed reduced deposition of IgG along the GBM, whereas
IgG deposition in the con-CD40-HN group was equivalent to
that in the HN group (Figure 5d).
con-CD40-HN
PAS
DEC-CD40-HN
HN CFA
Glomerulosclerosis Tubular atrophy
4
3
2
1
0
S
e
m
i
q
u
a
n
t
i
t
a
t
i
v
e

s
c
o
r
e
c
o
n
-
C
D
4
0
-
H
N
D
E
C
-
C
D
4
0
-
H
N
H
N
C
F
A
c
o
n
-
C
D
4
0
-
H
N
D
E
C
-
C
D
4
0
-
H
N
H
N
C
F
A
*
* **
**
**
*
*
4
3
2
1
0
S
e
m
i
q
u
a
n
t
i
a
t
i
v
e

s
c
o
r
e
Figure 4 | CD40 DNA vaccination reduces renal structural injury. Kidney tissues harvested at week 12 post Fx1A/complete Freunds
adjuvant (CFA) immunization were analyzed with periodic acidSchiff (PAS) staining. (a) Representative pictures from each group are shown
(magnification 200). (b) Semiquantitative scoring of glomerulosclerosis and tubular atrophy by a blinded observer.
25
Results are expressed as
mean

s.d. (*Po0.05; **Po0.01). HN, Heymann nephritis.

226 Kidney International (2013) 83, 223232
bas i c r es ear ch Y Wang et al.: DC-targeted CD40 DNA vaccine prevents HN
scDEC-CD40 DNA vaccination upregulates CTLA-4 mRNA
expression of draining lymph nodes
To determine the mechanism underlying the protective effects
of CD40 DNA vaccination, we examined the expression of
several cytokines and costimulatory molecules in draining
lymph nodes (DLNs) at week 12 post Fx1A/CFA immunization
by semiquantitative real-time PCR. CTLA-4 mRNA level was
upregulated in the DEC-CD40-HN group, and this was not
because of changes in CD40 mRNA, which remained un-
changed (Figure 6). No significant changes were detected for
other cytokines and costimulatory molecules (data not shown).
In vitro effects of serum antibody on B-cell activation and
T-cell proliferation
To determine whether the protective effects of scDEC-CD40
vaccination on HN are due to changes in B-cell activation, we
assessed the in vitro effects of serum on spleen cells from
normal Lewis rats. Sera from the DEC-CD40-HN or CFA
group collected at week 4 post Fx1A/CFA immunization were
used. For control purpose, an agonist monoclonal antibody
against CD40 was used simultaneously in this experiment.
B-cell activation was determined by CD86 expression. As
compared with agonist CD40 Ab, CD86 was not induced by
serum from DEC-CD40-HN and CFA rats (Figure 7a).
To determine whether anti-CD40 autoantibody, generated
as a result of CD40 vaccination, can inhibit T-cell prolifera-
tion by costimulatory blockade, we assessed in vitro effects of
serum on a mixed lymphocyte reaction. Carboxyfluorescein
diacetate succinamidyl ester (CFSE)labeled responder cells
were cocultured with stimulator cells (irradiated Wistar rat
lymphocytes) in the presence or absence of 5% serum. We
observed a significant reduction of CD8

T-cell proliferation
300
200
100
0
con-CD40-HN
DEC-CD40-HN
HN
CFA
* *
** **
CD4+ CD8+ Macrophage
I
m
m
u
n
e

c
e
l
l

i
n
f
i
l
t
r
a
t
i
o
n
s
N
o
.

o
f

c
e
l
l
s

p
e
r

x
2
0
0

f
i
e
l
d
con-CD40-HN DEC-CD40-HN
HN CFA
60
40
20
0
**
***
*
***
D
e
n
s
i
t
y
/
a
r
e
a
c
o
n
-
C
D
4
0
-
H
N
D
E
C
-
C
D
4
0
-
H
N
H
N
C
F
A
CD4
CD8
Macrophage
con-CD40-HN DEC-CD40-HN
HN CFA
con-CD40-HN DEC-CD40-HN
HN CFA
con-CD40-HN DEC-CD40-HN
HN CFA
Figure 5 | Infiltrating immune cells in the renal cortex and glomerular immunoglobulin G (IgG) deposition were reduced by scDEC-CD40
vaccination. Immunohistochemical staining was performed to assess the number of CD4

, CD8

, or CD68

(MF) cells in the renal cortex at week 12

post Fx1A/complete Freunds adjuvant (CFA) immunization, and IgG deposition was assessed by immunofluorescence staining. (a) The number of cells
in one 200 field is shown (average number counted from 10 of 200 random fields per animal). Values are presented as means

s.d. (*Po0.05;
**Po0.01). (b) Representative pictures of cells for each staining are shown (magnification 200). (c) Representative pictures of glomerular IgG staining
are shown (magnification 400). (d) Fluorescence intensity of glomerular IgG staining was quantified using Image J and expressed as density/area
(average number counted from 45 glomeruli of 400 field per group). Values are meanss.d. (*Po0.05; **Po0.01; ***Po0.001). HN, Heymann
nephritis.
Kidney International (2013) 83, 223232 227
Y Wang et al.: DC-targeted CD40 DNA vaccine prevents HN bas i c r es ear ch
in the DEC-CD40-HN serumtreated group compared with
the HN serum group (Figure 7b). A representative plot is
shown in Figure 7c.
In vivo assessment of B-cell depletion after CD40 vaccination
The protective effects of anti-CD40 therapy on allograft
survival in nonhuman primates have been associated with
B-cell depletion.
10
We therefore assessed B-cell numbers in
the spleens of all four groups. As shown in Figure 8a, B-cell
numbers were not significantly altered by CD40 vaccination.
Figure 8b shows representative immunohistochemical stain-
ing of B cells in each group.
DISCUSSION
Our current study demonstrates the therapeutic potential of
CD40 DNA vaccine in treating autoimmune renal disease.
Our results show that an induction of anti-CD40 antibody by
DNA vaccination is enhanced by DC targeting. This antibody
10
8
6
4
2
0
con-CD40-HN
DEC-CD40-HN
HN
CFA
*
R
e
l
a
t
i
v
e

m
R
N
A

e
x
p
r
e
s
s
i
o
n

(
L
N
)
CTLA-4 CD40
Figure 6 | CTLA-4 mRNA expression in draining lymph nodes
(DLNs) was upregulated by scDEC-CD40 vaccination, whereas
CD40 mRNA expression remained unchanged. Expression was
normalized against glyceraldehyde 3-phosphate dehydrogenase
(GAPDH). Results are expressed as mean

s.d. (*Po0.05). CFA,

complete Freunds adjuvant; HN, Heymann nephritis.
60
40
20
0
*
*
%

o
f

C
D
8
6
+

B

c
e
l
l
s
D
E
C
-
C
D
4
0
-
H
N

s
e
r
u
m
C
F
A

s
e
r
u
m
C
D
4
0

a
g
o
n
is
t
ic

A
b
CD8 CD4
50
40
30
20
10
0
con-CD40-HN
DEC-CD40-HN
HN
*
P
e
r
c
e
n
t
a
g
e

o
f
p
r
o
l
i
f
e
r
a
t
i
n
g

c
e
l
l
s
con-CD40-HN DEC-CD40-HN HN
CD4+gated
CD8+gated
250k
200k
150k
100k
50k
250k
200k
150k
100k
50k
S
S
C
-
A
11% 8%
24% 16% 36%
12%
0 10
2
10
3
10
4
10
5
0 10
2
10
3
10
4
10
5
0 10
2
10
3
10
4
10
5
CFSE
Figure 7| Serum containing anti-CD40 autoantibody suppressed B-cell activation and T-cell proliferation. (a) Splenocytes from normal
Lewis rats were cultured for 5 days in the presence of 10% serum from the DEC-CD40-HN or complete Freunds adjuvant (CFA) groups or with
1 mg/ml of anti-mouse/rat CD40 agonist antibody (n 3). Results are expressed as percentage of CD86 B cells (gated on live cell population
and B cells (OX33 )). (b) A statistical analysis of the percentage of proliferating cells (CD4

- or CD8

-gated cell population) is shown. Values

are meanss.d. (*Po0.05). (c) Carboxyfluorescein diacetate succinamidyl ester (CFSE)labeled Lewis T cells were stimulated with irradiated
Wistar lymphocytes for 5 days in the presence of 5% serum from the con-CD40-HN, DEC-CD40-HN and Heymann nephritis (HN) groups,
respectively (n 35). Results are expressed as percentage of proliferating cells (CD4

- or CD8

-gated cell population). Values are means

s.d.
(*Po0.05).
228 Kidney International (2013) 83, 223232
bas i c r es ear ch Y Wang et al.: DC-targeted CD40 DNA vaccine prevents HN
induction was persistent in the disease model. CD40
vaccination alone had some protective effect against protein-
uria but was exceedingly limited. However, by targeting the
CD40 to DCs, the effect was greatly enhanced, with a
prevention of the development of proteinuria, an improve-
ment in renal function, and a reduction in histological injury,
glomerular antibody deposition, and immune infiltration in
this group. As compared with other antibodies, the
antibodies generated by DNA vaccination were specific,
potent, and nondepleting.
Clinically, the costimulatory molecules have been an
obvious target for therapeutics with successful introduction
clinically of agents such as belatacept tailored to inhibit the
B7-CD28 pathway in clinical use in transplantation and other
areas.
23
Agents targeted at the CD40-CD154 pathway had
initially reached clinical use when targeted at CD154 but were
limited by side effects.
24
Newer agents targeting CD40 do not
seem to have the thrombotic risk of the previous agents,
which would be in keeping with the likely cause of
thrombosis in the CD154 studies being due to CD154
expressed on platelets.
13
Currently, CD40 blocking reagents
have been applied in large animal studies and seem much
more promising.
13,14
Thus, our studies appear in keeping
with the research demonstrating a key role of CD40-CD154
in immune activation in autoimmune disease and the success
in therapeutically blocking this pathway. The antibodies
generated by the modified vaccine appear to be blocking and
nondepleting, both of which are crucial clinically and may
potentially function at the site of T cell-DC/APC interaction
in the DLN where we find an increase in expression of
CTLA-4, a negative regulator of T-cell function. The efficacy
in larger animal models for human testing remains to be
studied.
Interestingly, despite no proteinuria, the CD40-DEC-205-
treated rats had a slightly lower level of serum albumin
compared with CFA-treated rats and a lower level of serum
creatinine, possibly suggesting some constitutional change
in the treated rats. An effect from the CD40 vaccine
alonetreated group was suggested by histology with a slight
improvement in proteinuria, although both cellular infiltra-
tion and immune deposits were not significantly improved in
this group.
In conclusion, CD40 appears to be an excellent therapeu-
tic target in autoimmune renal disease. The scDEC-CD40
vaccine was more effective compared with the scControl-
CD40 vaccine in attenuating progression of HN. This
suggests that DNA vaccination enhanced by DC targeting
can be used therapeutically to block specific immune
pathways that are involved in renal disease progression.
MATERIALS AND METHODS
Experimental animals
Inbred male Lewis rats (aged 6 weeks and weighing 180200 g),
inbred male Wistar rats, and outbred male SpragueDawley rats
(aged 8 weeks and weighing 200220 g) were purchased from the
Animal Resources Centre in Perth, Australia, and maintained under
standard sterile conditions in the Department of Animal Care at
Westmead Hospital. Experiments were carried out in accordance
with the protocols approved by the Animal Ethics Committee of
Sydney West Area Health Service.
Antibodies
Antibodies used in immunohistochemical studies included the
following: mouse anti-rat CD4 (OX35) (Serotec, Oxford, UK),
mouse anti-rat CD8a (OX8) (eBioscience, San Diego, CA), mouse
anti-rat CD68 (ED1) (Serotec), mouse anti-rat CD45RA (OX33)
(BD Pharmingen, San Diego, CA), and biotinylated goat anti-mouse
400
300
200
100
0
N
o

o
f

B

c
e
l
l
s

p
e
r

x
4
0
0

f
i
e
l
d
H
N
C
F
A
c
o
n
-
C
D
4
0
-
H
N
D
E
C
-
C
D
4
0
-
H
N
con-CD40-HN DEC-CD40-HN
HN CFA
Figure 8 | CD40 vaccination does not deplete B cells.
Immunohistochemical staining of B cells using OX33 was performed
on spleen sections collected at week 12 post Fx1A/complete Freunds
adjuvant (CFA) immunization. (a) The numbers of B cells (OX33 )
in one 400 field are shown (average number counted from three
of 400 random fields per animal). Values are means

s.d. (b)
Representative pictures demonstrate intact B-cell numbers and
splenic architecture (magnification 400). HN, Heymann nephritis.
Kidney International (2013) 83, 223232 229
Y Wang et al.: DC-targeted CD40 DNA vaccine prevents HN bas i c r es ear ch
immunoglobulin (Zymed Laboratories, San Francisco, CA). FITC-
goat anti-rat IgG used for immunofluorescence staining was from
Zymed Laboratories. APC-mouse anti-rat CD4 and PE-mouse anti-
rat CD8 used for flow cytometry staining were purchased from BD
Pharmingen. APC-mouse anti-rat CD45RA (OX33) used for flow
cytometry staining was from eBioscience. Anti-mouse/rat CD40
antibody (clone: HM40-3) (eBioscience) was used for in vitro
stimulation experiments. PE anti-mouse CD40 antibody (FGK45.5)
used for the binding assay was kindly provided by Dr Irina
Caminschi from Burnet Institute, Melbourne, Victoria, Australia.
Propidium iodide used for flow cytometry analyses was from Merck
Millipore Biosciences (Darmstadt, Germany).
Construction and modification of the CD40 DNA vaccine
pSC-DEC-OLLA and pSC-GL117-OLLA vectors were kindly pro-
vided by Dr Godwin Nchinda from the USA.
20
Mouse cDNA for
amplification of CD40 was reverse-transcribed from total RNA
extracted from the spleen of Adriamycin Nephropathy mouse. Total
RNA was extracted using an RNeasy Mini kit (Qiagen, Hilden,
Germany) and reverse transcription was performed using a
SuperScript III First-Strand Synthesis system (Invitrogen,
Carlsbad, CA). The gene encoding the extracellular domain of
CD40 was modified by fusing in frame to a sequence encoding a P30
epitope (FNNFTVSFWLRVPKVSASHLE,
17
), with P30 at the
C-terminus of CD40. Construction of CD40-P30 fusion DNA was
performed using sequence overlapping primer extension PCR with
specific primers: CD40-Nterm-FW: 5
0
-CGTGGCGGCCGCCTAGGG
CAGTGTGTTACGTGC-3
0
; CD40-P30-Rev: 5
0
-GGGCACGCGCAGCCA
GAAGCTGACGGTGAAGTTGTTGAATCGCATCCGGGACTTTAAA
CC-3
0
; P30-Cterm template: 5
0
-AGCTTCTGGCTGCGCGTGCC
CAAGGTCAGCGCCAGCCACCTGGAG-3
0
; and P30-Cterm-Rev:
5
0
-CAGTCCGCGGCTCCAGGTGGCTGGCGCTGAC-3
0
. The CD40-
P30 fusion DNA fragment was then cloned into pSC-DEC-OLLA or
pSC-GL117-OLLA vector to generate scDEC-CD40 or scControl-
CD40 DNA vaccines. The sequences of the two CD40 vaccines were
confirmed by DNA sequencing using specific primers: FW 5
0
-GCG
AATGAATTGGGACCT-3
0
and Rev 5
0
-CTTCTGAGATGAGTTTTTG
TTCG-3
0
. Plasmid DNA was prepared in large scale using Plasmid
Maxi Prep (Qiagen).
In vitro expression of DNA vaccines by western blot
Plasmid DNAs (scControl-CD40, scDEC-CD40, scControl vector,
and scDEC vector) were transiently transfected into HeLa cells, and
24 h post transfection total cell lysates were immunoblotted with
goat anti-mouse CD40, followed by donkey anti-goat Ig-horseradish
peroxidase, and detected with a chemiluminescent ECL detection
system (Chemicon, Temeculat, CA).
Binding of the scDEC-CD40 fusion protein to
DEC205-expressing CHO-K1 cells
Full-length mouse DEC-205 cDNA was expressed in CHO-K1 cells,
and DEC205-expressing CHO-K1 cells were sorted by flow
cytometry (CHO-DEC-205 transfectants). Parental CHO-K1 cells
or CHO-DEC-205 transfectants were incubated (30 min; 4 1C) with
supernatant from nontransfected cells (N/T sup) or with super-
natant from cells transfected with scControl-CD40 or scDEC-CD40
constructs. Cells were washed and stained with PE rat anti-CD40
mAb (FGK4.5). Propidium iodide (0.5 mg/ml) was added to the
final cell wash and cells were subjected to flow cytometry analyses.
Propidium iodidepositive dead cells were excluded from the
analyses.
DNA vaccination and induction of active HN
Rats were divided into four groups: the DEC-CD40-HN group
(n 5), comprising rats vaccinated with scDEC-CD40, followed by
Fx1A immunization; the con-CD40-HN group (n 5), comprising
rats vaccinated with scControl-CD40, followed by Fx1A immuniza-
tion; the HN group (n 4), comprising nonvaccinated rats with
Fx1A immunization; and the CFA group (n 3), comprising
nonvaccinated rats with CFA immunization. Rats were immunized
with DNA by i.m. injection in conjunction with electroporation in
the anterior tibialis (TA) muscles of the right hind leg. One week
before DNA immunization, rats were injected i.m. with 0.75%
bupivacaine (1 ml/g body wt; Sigma, St Louis, MO), which was
followed by two i.m. DNA injections (3 weeks apart with 300 mg/
150 ml sterile H
2
O/rat/injection) at the same location as the
bupivacaine injection. Each DNA injection was followed immedi-
ately by square wave electroporation at the injection site using
BTX830 two-needle array electrodes (BTX Harvard Apparatus, San
Diego, CA). The distance between the electrodes was 10 mm and the
array was inserted longitudinally relative to the muscle fibers. In vivo
electroporation parameters were as follows: 100 V/cm; 50-msec
pulse length; six pulses with reversal of polarity after three pulses.
Two weeks after the second DNA injection, HN was induced. To
induce HN, Lewis rats were immunized subcutaneously in both
hind footpads. Each footpad was injected with 100 ml of emulsion
containing 15 mg of Fx1A, 1 mg mycobacterium tuberculosis HRa37
(Difco, Detroit, MI), 100 ml of IFA (Sigma-Aldrich, St Louis, MO),
and 100 ml of PBS. The CFA control rats were immunized with
emulsion without Fx1A. Fx1A was extracted from outbred male
SpragueDawley rats as described previously.
16
All procedures were
performed with rats under isoflurane anesthesia.
Enzyme-linked immunosorbent assay
Serum anti-CD40 antibody level was evaluated using an ELISA
Ensemble kit for the detection of rat primary antibody (Alpha
Diagnostic International, San Antonio, TX) according to the
manufacturers protocols. C96 Maxisorp MicroWellt (NUNC,
Thermo Fisher Scientific, Waltham, MA) plates were used and
recombinant mouse CD40 protein (R&D system, Minneapolis, MN)
was used for coating of the plates at a concentration of 1 mg/ml in
100 ml of coating buffer. All sera were diluted 1:10 with a sample
diluent as provided in the kit. Absorbance was measured at 450 nm
with a Multiskan EX Microplate photometer (Thermo Fisher
Scientific). Absorbance at 570 nm was used as background correction.
Renal function
Blood and 16-h urine samples were collected every 2 weeks after the
induction of HN. Urine protein concentration was measured using a
colorimetric assay (Bio-Rad, Hercules, CA) based on the method of
Bradford. Urine creatinine, serum albumin, and serum creatinine
were analyzed using an automated chemistry analyzer VITROS
(Ortho Clinical Diagnostics, Johnson & Johnson, New Brunswick, NJ)
by staff at the Institute of Clinical Pathology and Medical Research
at Westmead Hospital.
Renal histology and immunohistochemistry
Coronal sections of the kidney were fixed in 10% neutral-buffered
formalin and embedded in paraffin. Sections of paraffin block
230 Kidney International (2013) 83, 223232
bas i c r es ear ch Y Wang et al.: DC-targeted CD40 DNA vaccine prevents HN
measuring 4 mm in thickness were stained with periodic acidSchiff s
reagent and counterstained with hematoxylin. Renal histopathology
was graded as previously described.
25
Immunohistochemical staining
was performed to determine the infiltrations of CD4 , CD8 T
cells, and macrophages in the kidney. For staining of CD4 T cells,
frozen sections (cut at 5 mm from kidney tissues embedded in OCT
compound) were used. Further, for staining of CD8 T cells and
macrophages, paraffin sections were used. Sections were incubated
with primary antibody (16 h, 4 1C), followed by secondary antibody
incubation (30 min, RT) as previously described.
25
Immune cell infiltration was quantified by counting 10
consecutive high-power fields per animal and expressed as cells
per 200 field. The number of OX33-positive B cells in the spleen
was quantified by counting three random high-power fields per
animal and expressed as cells per 400 field. Slides were scanned
using a ScanScope digital slide scanner (Aperio Technologies, Vista,
CA). Image analysis was performed using Image J software (NIH,
Bethesda, MD).
Immunofluorescence staining of IgG in kidney sections was
performed to assess IgG deposition on glomeruli. Frozen sections
were incubated with goat serum (15 min, RT), followed by FITC-
goat anti-rat IgG antibody (2 h, RT). Images (magnification 400)
were taken using a DeltaVision core microscope (Applied Precision,
Washington). Fluorescence density was quantified using Image J
software (NIH).
Mixed lymphocyte reaction
Lewis rat lymphocytes from the spleen were labeled with CFSE
according to the manufacturers protocol (Invitrogen). CFSE-labeled
Lewis lymphocytes (410
5
) (responder cells) were stimulated with
irradiated (25 Gy 137Cs) unlabeled Wistar lymphocytes (410
5
) in
flat-bottomed 96-well plates. Cells were cultured in RPMI1640
medium (Lonza, Basel, Switzerland) supplemented with 10% fetal
bovine serum, 100 m/ml penicillin/streptomycin, 10 mmol/l Hepes,
sodium pyruvate, nonessential amino acids (NEAA), 50 mmol/l
2-ME, and 2 mmol/l L-glutamine (Invitrogen) at 37 1C, 5% CO2.
After 5 days of culture, cells were harvested and subjected to flow
cytometry analysis.
Flow cytometry analysis
Single cell suspension was directly stained with APC- and PE-
conjugated mouse anti-rat mAb to cell surface antigens. All samples
were analyzed on a FACSCanto or FACSCalibur flow cytometer
(Becton Dickinson, Mountain View, CA). Flowjo software was used
for analysis (Tree Star, Ashland, Oregon).
Real-time PCR
Total RNA was isolated from rat DLNs and reverse transcribed (as
above). cDNA was subjected to quantitative PCR analysis using
Taqman Gene Expression Assays specific for the genes of interest
(Applied Biosystem, Melbourne, VIC, Australia). PCR reaction mix
was subjected to a Rotor-Gene 3000 thermal cycler (Corbett Life
Science, Brisbane, QLD, Australia) for acquisition and analysis.
Statistical analysis
Statistical analysis was performed using one-way analysis of variance
for multiple comparisons. For comparison between two groups, the
Student t-test was performed. Results are expressed as the group
mean

s.d. Differences were considered significant at Po0.05.

Statistical analysis was performed using Prism 5 (GraphPad Software,
La Jolla, CA).
DISCLOSURE
All the authors declared no competing interests.
ACKNOWLEDGMENTS
This work was supported by research grants from the National Health
and Medical Research Council of Australia (NHMRC grant 571343 and
632517). We thank the animal house staff in the Westmead Hospital
Animal House Facility for care of the animals. We also thank Godwin
Nchinda (The Rockefeller University) for kindly providing the DEC205
vectors and Virginia James (Westmead Millennium Institute, Australia)
for her technical assistance in histology.
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