Immunology and Cell Biology (2014) 92, 359367

& 2014 Australasian Society for Immunology Inc. All rights reserved 0818-9641/14
www.nature.com/icb

ORIGINAL ARTICLE

Induction of antigen-positive cell death by the

expression of Perforin, but not DTa, from a DNA
vaccine enhances the immune response
Tessa Gargett1,5, Branka Grubor-Bauk1, Tamsin J Garrod1, Wenbo Yu1, Darren Miller2, Lee Major3,
Steve Wesselingh4, Andreas Suhrbier3 and Eric J Gowans1
The failure of traditional protein-based vaccines to prevent infection by viruses such as HIV or hepatitis C highlights the need
for novel vaccine strategies. DNA vaccines have shown promise in small animal models, and are effective at generating antiviral T cell-mediated immune responses; however, they have proved to be poorly immunogenic in clinical trials. We propose that
the induction of necrosis will enhance the immune response to vaccine antigens encoded by DNA vaccines, as necrotic cells
are known to release a range of intracellular factors that lead to dendritic cell (DC) activation and enhanced cross-presentation
of antigen. Here we provide evidence that induction of cell death in DNA vaccine-targeted cells provides an adjuvant effect
following intradermal vaccination of mice; however, this enhancement of the immune response is dependent on both the
mechanism and timing of cell death after antigen expression. We report that a DNA vaccine encoding the cytolytic protein,
perforin, resulted in DC activation, enhanced broad and multifunctional CD8 T-cell responses to the HIV-1 antigen GAG and
reduced viral load following challenge with a chimeric virus, EcoHIV, compared with the canonical GAG DNA vaccine. This
effect was not observed for a DNA vaccine encoding an apoptosis-inducing toxin, DTa, or when the level of perforin expression
was increased to induce cell death sooner after vaccination. Thus, inducing lytic cell death following a threshold level of
expression of a viral antigen can improve the immunogenicity of DNA vaccines, whereas apoptotic cell death has an inhibitory
effect on the immune response.
Immunology and Cell Biology (2014) 92, 359367; doi:10.1038/icb.2013.93; published online 10 December 2013
Keywords: DNA vaccines; T cells; dendritic cells; cell death

In the global search for effective vaccines against major pathogens

such as HIV-1 and hepatitis C virus, the in vivo production of
neutralising antibodies has proved difficult to achieve and attention
has now focused on also inducing T cell-mediated immune responses.
It is thought that an early T-cell response at the site of infection may
be sufficient to clear infection or control virus replication.13 DNA
vaccines are known to induce cell-mediated immunity and humoral
immune responses, and result in antigen expression and processing in
a way that mimics viral protein production.46 DNA vaccines also
have practical advantages, as they are highly stable and more simply
and rapidly manufactured than protein or recombinant virus
vaccines,7,8 and do not induce vector-specific immunity that is an
issue for recombinant virus vaccines.9,10 Unfortunately, DNA vaccines
have failed to live up to their potential and have demonstrated poor
immunogenicity in clinical trials.11 At present, DNA vaccines are most
successful when combined with a live virus vector1214 in a

heterologous prime-boost protocol, resulting in improved T cellmediated responses in particular.15 Thus, any improvement in DNA
vaccine immunogenicity will likely also enhance the potency of these
heterologous prime-boost protocols.
Attempts to develop new vaccination strategies by improving DNA
vaccine immunogenicity have largely focused on the coexpression of
immunostimulatory proteins such as cytokines,16 costimulatory
molecules CD80 and CD8617 and Toll-like receptor ligands.18,19
Another recent approach has been to target antigen directly to
dendritic cells (DCs). As DCs are the only antigen-presenting
cells capable of cross-presenting exogenous antigen to naive CD8
T cells,20,21 they are an important target for a vaccine designed to
prime T cells against HIV-1. In the past, protein vaccines have been
conjugated to antibody that binds DC surface receptors such as
DEC205 and Clec9A.22,23 This strategy increased DC antigen
presentation and T-cell responses following protein vaccination that

1Virology Laboratory, Discipline of Surgery, University of Adelaide, Basil Hetzel Institute for Translational Medicine, Adelaide, South Australia, Australia; 2Experimental
Therapeutics Laboratory, University of South Australia, Adelaide, South Australia, Australia; 3Inflammation Biology Group, Queensland Institute of Medical Research, Brisbane,
Queensland, Australia and 4South Australia Health and Medical Research Institute, Adelaide, South Australia, Australia
5Current address: Experimental Therapeutics Laboratory, Hanson Institute, Royal Adelaide Hospital, Adelaide, South Australia, Australia.
Correspondence: Dr T Gargett, Experimental Therapeutics Laboratory, Level 4 Hanson Institute Building, Royal Adelaide Hospital, Frome Road, Adelaide, South
Australia 5000, Australia.
E-mail: tessa.gargett@adelaide.edu.au
Received 7 August 2013; revised 28 October 2013; accepted 9 November 2013; published online 10 December 2013

DNA vaccines to induce cell death

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has traditionally induced mainly humoral immune responses.

However, such approaches require additional adjuvants such as
polyinosinic:polycytidylic acid to ensure Toll-like receptor 3
stimulation and DC activation.24
Herein, we propose an alternative strategy to achieve DC targeting
of antigen and improved DNA vaccine immunogenicity by inducing
cell death to provide antigen for uptake by DCs, and adjuvant signals
for DC activation. Necrotic cell death is in particular known to
provide danger signals to the immune system via the release of
intracellular factors that act as damage-associated molecular patterns.25,26 The damage-associated molecular patterns such as uric
acid27 and high mobility group box 1 (HMGB-1)28 are capable
of signalling to DCs via diverse receptors. More recently, the
receptor Clec9A and its ligand, F-actin, have been identified
as a mechanism by which DCs sense necrosis and target antigen for
cross-presentation.29,30 Furthermore, necrosis also leads to NLRP3
inflammasome activation within cells and release of proinflammatory
cytokines interleukin (IL)-1B and IL-18.31 In addition, tissue damage
resulting in the release of double-stranded DNA into the extracellular
space has been identified as one of the underlying mechanisms of
Alum adjuvants.32 Our DNA vaccine was designed to encode an
antigen and a cytolytic protein that results in targeted killing of
antigen-positive cells. As a result, this vaccine will mimic a lytic virus
life cycle, and lytic viruses such as vaccinia are known to be highly
immunogenic.33,34
Suicide genes, which generally induce apoptosis of target cells, were
initially proposed for use as cancer therapies,35 but to date little
research has focused on suicide genes or cytolytic genes as potential
adjuvants for prophylactic vaccines. Previous studies have primarily
focused on proapoptotic genes such as Fas36 and caspases 2 and 3,37
or induced general tissue damage at the site of vaccination by
coadministration of a cytotoxic agent with a vaccine.3840 In
contrast, other groups have reported that the prevention of
apoptosis following DNA vaccination enhances the immune
response,41,42 indicating that attenuation of antigen expression and
death via the apoptotic pathway inhibits the immune response to
vaccine antigens. Thus, the role of apoptotic cell death in vaccination
is still controversial43 and the effect of targeted killing of antigenpositive cells via the necrotic or lytic pathway has not been reported.
The aim of this study was to investigate whether the induction of lytic
cell death after DNA vaccination by incorporating a cytolytic gene
into the vaccine has the potential to enhance antiviral immune
responses.
RESULTS
DTa and PRF cause cell death in vitro
DNA vaccines were constructed using the plasmid pVAX (Invitrogen,
Melbourne, VIC, Australia) that has been optimised for use as a DNA
vaccine,44 and encoded either as a luciferase (LUC) reporter gene or
the HIV-1 GAG gene under the control of the constitutive
cytomegalovirus (CMV) promoter. To investigate the effect of cell
death following DNA vaccination, bicistronic constructs were
designed to also encode either (1) perforin (PRF), an endogenous
mediator that disrupts cellular membranes and causes cell lysis,45,46 or
(2) diphtheria toxin subunit A (DTa), which inhibits protein synthesis
and causes apoptosis.47,48 These genes were inserted under the control
of the weaker SV40 promoter (Figure 1a). In our hands, the SV40
promoter resulted in 10-fold lower expression of luciferase in
HEK293T cells (Figure 1b) as compared with the CMV promoter,
similar to reports by others,49 and therefore permitted reduced levels
of PRF or DTa expression relative to the antigen expressed from the
Immunology and Cell Biology

CMV promoter. Additional constructs encoded a LUC-PRF or GAGPRF polyprotein under the control of the CMV promoter; the two
proteins were separated by the FMDV 2A protease to enable
autocleavage of the polyprotein.50 These additional constructs
allowed us to investigate the effects of the cytolytic gene, PRF, when
expressed at higher levels from the CMV promoter, as increased
concentrations of PRF within the cell were predicted to lead to more
effective cell killing and a potentially enhanced adjuvant effect.
Expression of the GAG antigen as well as PRF and DTa was also
confirmed by western blot that confirmed that 48 h after transfection
all DNA vaccines resulted in GAG expression (p55) and expression of
PRF or DTa (data not shown).
Expression of the LUC reporter, and the effects of PRF and DTa on
the kinetics of this expression, were determined by an in vitro
luciferase assay (Figure 1b). This revealed that, following transfection
with CMV-LUC-SV40-PRF, luminescence was equivalent to that
observed for cells transfected with the CMV-LUC control at 24 h
post transfection, but decreased from 48 h post transfection, most
likely indicating killing of luciferase-positive cells. CMV-LUC-SV40DTa-transfected cells showed lower relative luminescence at all time
points as compared with the CMV-LUC control, as did CMV-LUC2A-PRF-transfected cells.
To confirm that the loss of luciferase expression was a result of the
induction of cell death, the DNA vaccine constructs were also assessed
for their ability to induce cell death directly in vitro. After transfection
with the bicistronic plasmids encoding PRF or DTa, HEK293T cells
showed visible signs of cell death 48 h later by light microscopy
(Figure 1c). Flow cytometry was then performed to confirm cell
death, by Annexin V and propidium iodide (PI) staining to identify
apoptotic and necrotic cells, as described previously51,52 (Figure 1d).
At 48 h post transfection with CMV-LUC-SV40-PRF or CMV-LUCSV40-DTa, a significant proportion of cells was shown to be dead as
determined by forward and side scatter characteristics (38.2% and
43.2% respectively), compared with cells transfected with CMV-LUC
alone (15.5%). When PRF was encoded under the control of the
stronger CMV promoter (CMV-LUC-2A-PRF) cell death was greater
than that seen with CMV-LUC-SV40-PRF, with 48.9% of cells dead by
48 h. This confirmed that the reduction in luminescence observed in
the luciferase assay corresponded with the induction of cell death.
Within these dead cell populations, the proportion of apoptotic and
necrotic cells varied, reflecting the different mechanisms by which
DTa and PRF induced cell death. Transfection with CMV-LUC-SV40PRF or CMV-LUC-2A-PRF resulted in a higher proportion of
propidium iodide-positive necrotic cells than that detected following
transfection with CMV-LUC-SV40-DTa.
Live imaging of luciferase allows tracking of antigen expression
after intradermal vaccination
To investigate the effects of cell death on antigen expression following
DNA vaccination in vivo, LUC was used as a model antigen. This
allowed live imaging of the mice and tracking of antigen expression
after vaccination, and provides an indirect measure of cell death or
clearance resulting in a loss of luminescence. Female C57BL/6 mice
were vaccinated with a single dose of 50 mg DNA via the intradermal
(ID) route (ear pinnae dermis) and imaged periodically for luminescence. The inclusion of the PRF or DTa gene changed the kinetics of
luciferase expression after vaccination (Figures 2a and b). Mice that
received CMV-LUC demonstrated detectable luminescence to day 35
post vaccination, although the level of luminescence dropped E100fold over this period, indicating immune-mediated clearance of
luciferase-positive cells.53 In contrast, mice vaccinated with CMV-

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a
CMV

LUC or GAG

CMV

LUC or GAG

SV40

PRF

CMV

LUC or GAG

SV40

DTa

CMV

LUC or GAG

2A

CMV LUC

CMV LUC SV40 PRF

PRF

b
CMV LUC SV40 DTA

CMV LUC 2A PRF

CMV LUC

CMV LUC SV40 PRF

CMV LUC SV40 DTa

CMV LUC 2A PRF

Figure 1 DNA vaccines encoding PRF or DTa induce cell death in vitro. (a) Schematic diagram of the bicistronic DNA vaccine constructs. All vaccines use
the pVAX backbone, with a gene encoding an antigen (HIV-1 GAG or firefly luciferase (LUC)) under the control of the CMV promoter and PRF or DTa under
the control of the SV40 promoter. An additional construct encodes the antigen and PRF as a polyprotein controlled by the CMV promoter that self-cleaves at
the FMDV 2A protease sequence. (b) Luciferase expression following DNA vaccine transfection was confirmed by luciferase assay, and death of luciferasepositive cells was identified by loss of luminescence over time. Graphs show means.e.m. (c) Phase microscopy images (  10) reveal rounded, detached
HEK293T cells at 48 h post transfection with DNA vaccines encoding DTa or PRF. (d) Flow cytometry to identify dead cells, and Annexin V and propidium
iodide (PI) staining to identify apoptosis (Annexin V ) and necrosis (Annexin V PI , Annexin V  PI ) 48 h post transfection.

LUC-SV40-PRF showed a decrease in luminescence that was

undetectable by day 35. Close analysis of Figure 2 shows that the
rate of loss of luminescence (cell death) was more rapid from days
2035 in the CMV-LUC-SV40-PRF-vaccinated animals as compared
with the CMV-LUC-vaccinated animals. Thus, cell death was apparent
in the former animals from day 20 onwards.
Interestingly, the kinetics of luminescence were markedly different
to that observed in vitro (Figure 2), as a decrease occurred much later.
This highlights differences between the complex nature of in vivo
experiments and experiments performed in vitro in cultured cells.
Figure 2 shows that the luminescence values attained saturation and
essentially reached a plateau between days 0 and 20 and thereafter
showed a rapid decrease, particularly in the mice injected with CMVLUC-SV40-PRF as compared with CMV-LUC-injected mice. As
intradermal vaccination is likely to target different cell populations
that may show differential rates of cell death, the luminescence values
would be unable to detect any cell death until the level of expression
dropped below the level of saturation.
The CMV-LUC-SV40-DTa and CMV-LUC-2A-PRF groups showed
the most rapid decrease in luminescence, and no detectable luminescence was seen in these mice after day 14 (Figure 2b). In addition, the
CMV-LUC-SV40-DTa- and CMV-LUC-2A-PRF-vaccinated groups
demonstrated a 10-fold lower luminescence from day 1 after
vaccination, indicating that there was reduced antigen expression in
the dermis even at the earliest time point. Thus, the level of luciferase
expression varied following expression of DTa or PRF in vaccinated
mice, and also varied following PRF expression driven by the CMV or
SV40 promoters.

All mice that received the ID vaccination showed some irritation

(redness and swelling) around the site of injection; however this was
observed even for mice that received an injection of saline, and
rapidly subsided by days 23 post vaccination. No tissue damage was
observed macroscopically or microscopically by histological analysis
in any vaccinated group (Supplementary Figure 1). Thus, coexpression of DTa or PRF led to targeted killing of luciferase-positive cells
without excessive inflammation or tissue damage.
PRF enhances the T cell-mediated immune responses to HIV-1
GAG DNA vaccines
To investigate the effect of PRF and DTa on the immune response to a
viral antigen that contains multiple T-cell epitopes, we replaced the
luciferase reporter gene with a codon-optimised Clade B HIV-1 GAG
gene. Mice received 3 doses of 50 mg of CMV-GAG, CMV-GAG-SV40PRF, CMV-GAG-SV40-DTa or CMV-GAG-2A-PRF DNA via the ID
route at 4-weekly intervals, and the immune responses were assayed
10 days post final vaccination. Interferon-g (IFNg) enzyme-linked
immunosorbent spots (ELISPOTs) were performed using four overlapping peptide pools (E30 15-mer peptides per pool) representing
the complete GAG sequence, and an additional pool containing only
immunodominant peptides (which are otherwise present in pools 2
and 3). Mice vaccinated with CMV-GAG showed responses to all
peptide pools ranging from 50 to 1200 mean spot-forming units
(SFU; Figures 3ad). Mice vaccinated with CMV-GAG-SV40-PRF
showed a similar range of responses to pools 13 as control CMVGAG-vaccinated mice, but presented a 2.5-fold higher response to
peptide pool 4 as compared with CMV-GAG-vaccinated mice (mean
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362

d28

Pool 1

Pool 2
SFU/10^6 cells

200
150
100
50

1000

500

pV
AX
C
e
M
V
C mp
C GA MV ty
M
V G S GA
G
A V40 G
C G
M
SV DT
V
G 40 a
AG P
R
2A F
PR
F

V
M
C

AG

Pool 3

200
100

AG
G

F
PR
F
2A

Ta

40

SV
V

PR

pt

em

M
G
V

M
C

M
C

V
M

AG

pV

AX

G
AG
G

V
C

AG

S
AG V40
SV DT
V
G 40 a
AG P
R
2A F
PR
F

AG

y
pt
em

M
C

AX

1.0x1006

y
AG

pVAX empty
CMV LUC
CMV LUC SV40 PRF
CMV LUC SV40 DTA
CMV LUC 2A PRF

pV

Pool 5
SFU/10^6 cells

1.0x1005
1.0x1004

2000
1500
1000
500

SFU 155 vs 62, P 0.03) (Figure 3d). As this pool contains none of
the immunodominant GAG peptides, this represents a broadening of
the immune response to nondominant epitopes.
In contrast, the CMV-GAG-SV40-DTa-vaccinated mice showed
fivefold lower IFNg responses to peptide pool 3 (mean SFU 142 vs
696, P 0.003) and to the immunodominant peptides in pool 5
(mean SFU 272 vs 1270, P 0.001) as compared with CMV-GAGvaccinated mice (Figures 3c and e). Surprisingly, mice vaccinated with
CMV-GAG-2A-PRF showed no significant changes in IFNg responses
compared with CMV-GAG, despite the accelerated cell death observed
for this construct in the in vitro and in vivo assays.
Intracellular cytokine staining demonstrated that individual CD4
and CD8 T cells responded to vaccination by producing IFNg, tumor
necrosis factor-a (TNFa) and IL-2 (Figure 4 and Supplementary
Figure 2). Mice vaccinated with CMV-GAG-SV40-PRF showed
twofold higher levels of TNFa and IFNg-secreting CD8 T cells as
compared with mice vaccinated with CMV-GAG (mean percentage
0.21 vs 0.1, P 0.03; Figure 4a). There was a trend showing an
Immunology and Cell Biology

40

SV

AG

G
V

SV

AG
G
C

pt

AG
G

em

AX

C
V

Figure 2 In vivo imaging of luciferase. Live imaging of luciferase allows

antigen expression from DNA vaccines to be tracked in vivo.
(a) Representative IVIS images of luminescence in the mouse dermis at
days 7, 14 and 28 after ID vaccination with 50 mg CMV-LUCPRF or DTa.
(b) The expression of LUC after ID vaccination as determined by
quantification of luminescence in vaccinated mice. Vaccinations were
performed on groups of five female C57BL/6 mice. Graphs show
means.e.m.

40

10
20
30
Days post vaccination

D
Ta
4
AG 0 P
R
2A F
PR
F

1.0x1003

*p=0.001

pV

Luminescence (Log 10 total flux p/s)

500

300

SFU/10^6 cells

1000

CMV LUC 2A
PRF

1.0x1007

SFU/10^6 cells

*p=0.003

1500

CMV LUC PRF

Pool 4
*p=0.03

CMV LUC DTA

pV
AX

CMV LUC

em

pt
y

A
S
AG V4 G
0
D
V SV4 Ta
G
AG 0 P
R
2A F
PR
F

SFU/10^6 cells

250

40

d14

SV

d7

d28

d14

d7

Figure 3 IFNg ELISPOT to detect GAG-specific T-cell responses. Mice were

vaccinated ID with 3 doses of 50 mg DNA, 4 weeks apart, and spleens
harvested on day 10 after the final vaccination. Splenocytes were
restimulated with 4 different pools of overlapping peptides covering the
complete GAG protein (pools 14 shown in ad respectively), or a final pool
of C57BL/6 MHC I- and II-restricted immunodominant peptides (e).
Vaccinations were performed on groups of eight female C57BL/6 mice.
Graphs show the mean SFU per 106 splenocytes (s.e.m.) minus the
background SFU counted for unstimulated control wells.

increase in rare, multifunctional CD8 T cells that secreted TNFa, IFNg

and IL-2 simultaneously (mean percentage 0.078 vs 0.04) in mice
vaccinated with CMV-GAG-SV40-PRF when compared with CMVGAG (Figure 4b). All vaccinated groups (except mock-vaccinated)
produced CD4 T cells that secreted multiple cytokines in response to
GAG immunodominant peptides but no differences were observed
between groups (Figures 4c and d). Consistent with the ELISPOT
data described above, the CMV-GAG-2A-PRF vaccination induced no
significant changes in CD8 and/or CD4 T-cell cytokine production
compared with CMV-GAG.
Thus, only PRF increased the immune response to the GAG
peptide pools, and only when expressed from the weaker SV40
promoter (CMV-GAG-SV40-PRF). An increased level of expression of

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T Gargett et al
363

3
2
1
0

80 p=0.029

60
50
40

PR

Ta

AG

40
SV

G
V
M

40
35

Ta
D
SV

40

40
SV
G

AG

AG
C

V
M

PR

AG
G

V
M

C
G

AX

em

PR

pt

30

p=0.03

45

pV

SV

SV

50

AG

AG

C
G

40

40

G
V
M

AG

Ta

0
pt

CD80+ Clec9A+ CD8a+ DC

in the draining lymph nodes
% of CD3e- CD11c+
MHCII+ DC

G
G
V

M
C

M
C

10

V
AG

pV

G
V

15

em

AX

40

em

PR

pt

Ta
AG

AG
G

SV

SV

pV

M
C

% of CD3e- CD11c+
MHCII+ DC

25 p=0.057
20

AX

PRF increases the frequency of cross-presenting CD8a DCs

The data presented above showed that expression of the cytolytic
protein PRF and the apoptotic protein DTa resulted in opposite
effects on the T cell-mediated immune response to GAG. As DCs are
crucial to the activation of naive T cells in secondary lymphoid
organs,55,56 we wished to determine whether there was an increase in
the frequency of activated DCs present in the auricular draining
lymph node as a result of the inclusion of PRF in the vaccine that may
have contributed to the improved T-cell immune response reported
above. To determine whether PRF- and DTa-induced cell death
resulted in increased frequency of activated DCs, the auricular
draining lymph nodes were collected at different time points after a
single, 50 mg ID DNA vaccination. DC subpopulations were identified
as CD3e , CD11c MHCII and CD8a /  or Clec9A / 
(Figure 5 and Supplementary Figure 3). On day 14 post vaccination,
a significant increase in the frequency of CD11c DCs and cross-

15

CD80+ CD8a+ DC in the

draining lymph nodes

pV

PRF from the CMV promoter (CMV-GAG-2A-PRF) had no effect on

the immune response, and CMV-GAG-SV40-DTa actively decreased
the immune response when compared with the control CMV-GAG.
The inhibitory effect of DTa was likely due to a reduction in antigen
levels caused by the ability of DTa to inhibit de novo protein synthesis
and kill cells via apoptosis, which is known to be a less inflammatory
form of cell death than necrosis.54

40

G
V
M

AX

em

pt

AG

20

40

25

SV

AG

% of CD3e- CD11c+
MHCII+ DC

8 p=0.057

CD80+ DC in the
draining lymph nodes
30 p=0.029
*

G
AG

pV

AX

em

SV
G
AG 40
D
Ta
SV
40
PR
F

em

AX
pV

% of CD3e- CD11c+
MHCII+ DCC

Clec9A+ CD8a+ DC in
the draining lymph nodes

Figure 4 Multifunctional T cells detected by flow cytometry. Intracellular

cytokine staining for GAG-specific T-cell responses after DNA vaccination, as
described in the legend for Figure 3. Splenocytes were restimulated for 12 h
with the immunodominant peptide pool in the presence of Brefeldin A.
(a) The percentage of IFNg- and TNFa-secreting CD8 T cells and (b) the
percentage of IFNg-, TNFa- and IL-2-secreting CD8 T cells in response to
GAG peptide stimulation. (c) Percentage of CD4 T cells secreting IFNg and
TNFa, and (d) IFNg, TNFa and IL-2 in response to GAG peptides.
Vaccinations were performed on groups of eight female C57BL/6 mice.
Graphs show the mean percentage of cytokine-secreting cells (s.e.m.),
minus the background percentage of staining for unstimulated control cells.

70

pt
y
M
G
AG V
G
C
M
A
SV
G
V
40
G
AG
D
T
SV
a
40
PR
F

% of CD3e- CD11c+
MHCII+ DC

4 p=0.028

CD8a+ DC in the
draining lymph nodes

pt
y
G
AG

% of CD3e- viable
cells

CD11c+ MHCII+ DC in the

draining lymph nodes

Figure 5 Detection of activated DCs. DCs in the draining lymph node are
activated after a single 50 mg DNA vaccination. Auricular lymph nodes were
harvested on days 3, 7, 10 and 14 post ID vaccination, and cells were
counted and stained for DC markers. All lymph nodes had equivalent
cellularity. All graphs represent data for day 14. CD3 , CD11c MHCII
cells were identified and assessed for CD8a, Clec9A and CD80 expression.
(a) Percentage of CD11c MHCII DCs. (b) Percentage of CD11c
MHCII CD8a DCs. (c) Percentage of CD11c MHCII CD8a Clec9A
DCs. The activation status of CD11c CD8 DCs as measured by CD80
expression on (d) CD11c MHCII DCs, (e) CD11c MHCII CD8a DCs
and (f) CD11c MHCII CD8a Clec9A DCs. DNA vaccinations were
performed on groups of four female C57BL/6 mice. Graphs show mean
percentage of cells (s.e.m.).

presenting CD8a DCs was detected in the draining lymph node of

mice vaccinated with CMV-GAG-SV40-PRF, but not CMV-GAGSV40-DTa, as compared with mice vaccinated with CMV-GAG (mean
percentage 3.2 vs 2.11 P 0.028 and 64.2 vs 53.7, P 0.029;
Figures 5a and b). At this time point there was also a trend towards
an increased frequency of Clec9A DCs in the CMV-GAG-SV40PRF-vaccinated mice compared with CMV-GAG (mean percentage
5.33 vs 44.6, P 0.057057; Figure 5c). This DC subset is capable of
sensing necrotic cells and targeting antigen for cross-presentation.29,30
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SV40-PRF, but not CMV-GAG, reduced the viral load (2.5-fold,

P 0.02) compared with that observed in mock-vaccinated mice
(Figure 6b). Likewise, in the peripheral blood, the viral load was
reduced by CMV-GAG-SV40-PRF (2.5-fold, P 0.01) but not by
CMV-GAG vaccination (Figure 6c). Thus, the immune response
induced by DNA vaccination containing the cytolytic gene, PRF,
resulted in improved control of viral replication at the site of
infection, and systemically, in EcoHIV-challenged mice.

The activation status of the DCs from the draining lymph node, as
measured by CD80 expression, was also determined. Vaccination with
CMV-GAG-SV40-PRF, but not with CMV-GAG-SV40-DTa, induced
a significant upregulation of CD80 on CD11c DCs and a trend
towards upregulation of CD80 on CD8a DCs, above that observed
after vaccination with CMV-GAG (mean percentage 2626.2 vs
2121.1%, P 0.02929, and 1818.8 vs 1414.5%, P 0.05757; Figures
5d and e). The Clec9A DCs were already highly activated in the
mock-vaccinated, CMV-GAG and CMV-GAG-SV40-PRF groups
with CD80 expression of 4050% of cells, respectively (Figure 5f),
although CD80 expression was significantly decreased in this
subset for mice vaccinated with CMV-GAG-SV40-DTa as compared
with CMV-GAG (mean percentage 44.9 vs 37.2, P 0.03). This
indicates that DTa expression, despite inducing cell death, had no
effect on DC population numbers, and decreased DC activation in
one subset. Thus, DTa and PRF induced different effects on DC
subpopulations after vaccination, and only PRF led to an increase in
the frequency of cross-presenting DCs, as well as an increase in
activation markers, consistent with necrosis-enhanced cross-presentation of antigen.

ns
0.00015
0.00010
0.00005

R
F

-P

AG

y
pt

AG

SV

40

VC
M
VG

C
M

X
pV
A

rm

al

em

ou
s

0.00000

no

SV

* p= 0.01

0.00020

R
F

AG
G
V-

M
C

Peripheral Blood

V-

VM
C

40
-P

y
pt
em

AX

ou
s

0.0000
m

F
R

0.0001

AG

AG

SV

40
-P

G
AG

pt
M

V-

em
AX

0.0002

no

rm

al

ou

se

0.0000

ns
0.0003

pV

0.0005

* p= 0.02

0.0004

rm
al

0.0010

Spleen

no

**p= 0.001
* p= 0.02

0.0015

EcoHIV mean normalised expression

(relative to RPL13a mRNA levels)

PEC

pV

EcoHIV mean normalised expression

(relative to RPL13a mRNA levels)

PRF DNA vaccination reduces the EcoHIV viral load after challenge
As noted above, enhanced cytokine production by T cells and
increased DC activation were observed after vaccination with DNAencoding HIV-1 GAG and the cytolytic protein, PRF. To determine
whether these changes represent enhanced immunity to viral infection, vaccinated mice were challenged with EcoHIV, a chimeric HIV
in which the HIV gp120 was replaced with gp80 from ecotropic
murine leukemia virus.57 EcoHIV encodes all other HIV proteins,
including GAG, from the HIV-1 Clade B NL4-3 strain. Mice received
two doses of 50 mg CMV-GAG or CMV-GAG-SV40-PRF DNA via the
ID route and were then challenged 10 days later with EcoHIV at a
dose of 1.5 mg p24 by the intraperitoneal route, as described
previously.58,59 Mice were culled 7 days after challenge and tissues
were collected to determine the viral load (Figure 6). Vaccination with
CMV-GAG reduced the viral load in the peritoneal exudate cells
below that detected in mock-vaccinated mice (twofold reduction in
normalised mRNA levels, P 0.02), whereas vaccination with CMVGAG-SV40-PRF resulted in a further decrease (fivefold reduction,
P 0.001; Figure 6a). In the spleen, vaccination with CMV-GAG-

EcoHIV mean normalised expression

(relative to RPL13a mRNA levels)

DISCUSSION
DCs are the only antigen-presenting cells capable of stimulating naive
CD8 T cells, and are therefore the target of many vaccine strategies
such as the direct targeting of antigen to DCs by conjugation with
monoclonal antibodies specific for DC receptors.22,23 In this study we
have taken a different approach to DC targeting and instead sought to
identify a method to enhance T-cell responses after vaccination by
inducing cellular necrosis, that is known to promote antigen uptake
and cross-presentation and activate DCs. We identified candidate
proteins perforin and DTa and tested their ability to induce
inflammatory cell death in vivo. Surprisingly, we found that
although DTa was more potent at inducing cell death in vivo, it
inhibited elements of the antigen-specific immune response. In
contrast, vaccination with a perforin-encoding DNA vaccine, which
induced cell death later after vaccination, enhanced the subsequent
immune response and reduced the viral load in EcoHIV-challenged
mice. This suggests that the timing of cell death and the level of
antigen expression are linked. Consistent with this insight, accelerated
cell death and reduced antigen expression after vaccination with
CMV-2A-PRF constructs (Figures 1 and 2) also failed to enhance the
antigen-specific immune response (Figures 3 and 4). This finding was
unexpected as we hypothesised that increased lytic cell death would
provide a more potent adjuvant effect and enhance the antigenspecific immune response.
As shown by our data, direct visualisation of antigen expression by
live imaging technology after DNA vaccination can provide valuable
information on the kinetics of antigen expression and insight into
factors that influence the development of an antigen-specific immune
response. The necessity for a threshold level and a minimum period
of antigen expression has been suggested by others in adenovirus and
DNA vaccine models,60,61 and we conclude that the mechanism and

Figure 6 EcoHIV challenge experiment. EcoHIV virus challenge after DNA vaccination with 2 doses of 50 mg CMV-GAG, CMV-GAG-SV40-PRF or pVAX
empty, 4 weeks apart. The mice were challenged 10 days later with EcoHIV (1.5 mg p24/mouse) and the viral load determined 7 days later. EcoHIV mRNA
levels in (a) peritoneal exudate cells (PECs), (b) spleen and (c) blood. Vaccinations were performed on groups of 8 female C57BL/6 mice. Graphs show
mean expression normalised to RPL13a (s.e.m.).
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DNA vaccines to induce cell death

T Gargett et al
365

kinetics of antigen-positive cell death also influenced the subsequent

immune response in our model system.
Inflammation can be potentially damaging;62 however, our strategy
induced no more tissue damage than standard ID injection protocols,
likely because cell death is restricted to cells that were transfected by
the DNA vaccine. This adjuvant system therefore has advantages over
more general mechanisms of inducing cell death at the site of
vaccination, such as the use of Alum adjuvants32 or electroporation,63,64 as it limits tissue damage and more closely mimics
the effects of lytic viruses, including live attenuated virus vaccines.
Despite a lack of inflammatory tissue damage, vaccination with the
perforin-encoding vaccine resulted in an increased frequency of
activated CD11c CD8a DCs in the draining lymph nodes, a cell
population that is essential for cross-presentation of antigen obtained
from dying cells to naive CD8 T cells (Figure 5). The T cell-mediated
immune response following vaccination was also modestly enhanced
by the coexpression of perforin, as determined by increased numbers
of memory T cells that secreted IFNg in response to stimulation by
GAG peptides (Figure 3). Although the CMV-GAG vaccine induced
strong GAG-specific, IFNg responses when administered via the ID
route, the addition of perforin broadened the immune response by
enhancing T-cell responses to nondominant epitopes. Perforin also
increased the frequency of CD8 T cells capable of secreting multiple
cytokines, a T-cell subset shown to be important in control of HIV-1
infection (Figure 4).65,66
Conversely, DTa failed to activate DCs or enhance immune
responses, but instead reduced the immune response to some GAG
epitopes. DTa inhibits protein synthesis and results in apoptosis that
is known to be less inflammatory than necrotic cell death.67
Viral antigen-positive cells that become apoptotic and are cleared
before they can undergo secondary necrosis will limit DC activation
and cross-presentation of antigen.25 This finding indicates that
although other cytolytic proteins may also be useful as vaccine
adjuvants, particularly if their expression results in lytic cell
death at a time point after the threshold of antigen expression
necessary to achieve memory immune responses is achieved,
apoptosis-inducing proteins should potentially be avoided as they
may have an inhibitory rather than an adjuvant effect. This result is
contrary to other studies that report improved vaccine efficacy
following the inclusion of a proapoptotic molecule,68,69 but in
agreement with other reports finding that necrosis but not
apoptosis is immunogenic.25,67
It has been established in both clinical and animal trials that IFNg
ELISPOT and intracellular cytokine staining of cells from spleen or
peripheral blood do not always correlate with protection from a viral
challenge.7072 Animal challenge models for HIV-1 are limited;
however, the EcoHIV chimeric virus provides a means to assess
vaccine candidates in a small-animal model and gain an initial
understanding of potential efficacy. Here we report that a DNA
vaccine encoding an antigen and a cytolytic protein elicits a higher
level of protective immunity as determined by viral challenge than a
DNA vaccine that only encodes the antigen, not only at the site of
infection in the peritoneal space, but also systemically in the spleen
and peripheral blood.
Thus, our data demonstrate that using a cytolytic protein to induce
death of viral antigen-positive cells resulted in increased dendritic cell
activation without excessive inflammation, and enhanced protection
from a viral challenge. This indicates that DNA vaccines have the
potential to be further optimised for use, either solely or as part of a
heterologous prime-boost protocol, in large animal models and
humans.

METHODS
DNA vaccines
All DNA vaccine constructs are based on the pVAX plasmid backbone
(Invitrogen). Genes for Firefly luciferase2 (Promega, Sydney, NSW, Australia)
or codon-optimised HIV-1 gag (clade B, a kind gift from Don Anson,
Womens and Childrens Hospital, Adelaide, SA, Australia) were inserted
downstream of the CMV promoter. An additional promoter, the SV40
promoter, and a poly-adenylation sequence were inserted into the pVAX
backbone and cytolytic or apoptotic gene candidates were inserted downstream of this second promoter. The mouse perforin clone was a kind gift from
Ilia Voskoboinik, Peter MacCallum Cancer Research Centre (Melbourne,
Australia). The DTa clone was obtained from Addgene (Cambridge, MA,
USA) (plasmid 13440, deposited by Phillipe Soriano73). All DNA vaccines were
purified using the Qiagen (Doncaster, VIC, Australia) Endotoxin-free Mega kit.

Cell culture
HEK293T cells (human embryonic kidney cell line) were grown in Dulbeccos
modified Eagles medium supplemented with 10% fetal calf serum and 1%
penicillin/streptomycin, and transfected with DNA using Fugene 6 reagent
(Promega). Cell morphology post transfection was assessed by light microscopy (Nikon, Sydney, NSW, Australia; Eclipse TE2000-U). Cell viability was
determined by Trypan blue staining.

In vitro expression and cell death assays

To assess luciferase expression and luciferase-positive cell death in vitro,
transfected cells and supernatants were transferred to opaque flat-bottom
96-well plates (Sigma, Castle Hill, NSW, Australia) and luciferase assays were
performed using 150 mg ml 1 D-Luciferin-K salt substrate (Perkin Elmer,
Melbourne, VIC, Australia) and analysed by FLUOStar Optima plate reader
(BMG Labtech, Mornington, VIC, Australia).
Luciferase assays to detect loss of reporter gene expression were performed
as described above, and cells were analysed at 24, 48 and 72 h. To identify the
mechanism of PRF- and DTa-induced cell death, markers of apoptosis and
secondary necrosis were assayed by flow cytometry using Annexin-V (Invitrogen) and propidium iodide (BD Biosciences, North Ryde, NSW, Australia) at
24, 48 and 72 h. Cells were analysed on a BD FACS Canto using the gating
strategy described in the relevant figures and results analysed with FlowJo
software (Ashland, OR, USA).

Mice
All experiments were approved by the University of Adelaide and the Womens
and Childrens Health Network animal ethics committees. C57BL/6 mice from
the University of Adelaide Laboratory Animal Services were maintained under
PC2 conditions and vaccinated at 68 weeks. All interventions were performed
under Domitor/Ketamine anaesthetic injected intraperitoneally and the
anaesthetic was reversed with intraperitoneal Antisedan (Zoetis, West Ryde,
NSW, Australia). Blood samples were obtained by retro-orbital bleed with
haematocrit tubes. Live imaging was performed on isofluorane-sedated
animals using an IVIS live imager (Perkin Elmer) after injection of D-luciferin
K 3 mg per 20 g mouse.

Immunisation
C57BL/6 mice received 50 ml of DNA (50 mg in saline) injected into the dermal
layer of the ear (ID injection, 25 ml per ear). Unless otherwise stated, mice
received a total of three doses of vaccine at 4-week intervals, and peripheral
blood samples were taken before each vaccination. At 10 days post final
vaccination, the mice were anaesthetised and killed, and spleen, peripheral
blood and draining lymph nodes collected.

ELISPOT
IFNg ELISPOT was performed on red blood cell-depleted splenocytes that
were restimulated with 2 mg ml 1 15-mer, 11 amino acid overlapping
gag peptide pools (NIH AIDS reagent bank 8117, Germantown, MD, USA)
or major histocompatibility complex (MHC) I- and II-restricted immunodominant peptides (listed in the HIV molecular immunology database,
Immunology and Cell Biology

DNA vaccines to induce cell death

T Gargett et al
366
http://www.hiv.lanl.gov/). Multiscreen-IP HTS plates (Millipore, Kilsyth, VIC,
Australia) were coated with anti-mouse IFNg (clone AN18, MabTech,
Thomastown, VIC, Australia) and secreted IFNg was detected with antimouse IFNg -biotin (clone R4-6A2, MabTech), streptavidin-AP (Sigma) and
SigmaFast BCIP/NBT.

Flow cytometry
Multi-colour intracellular cytokine staining was performed on splenocytes restimulated with immuno-dominant gag peptides for 12 h in the presence of
Brefeldin A. Staining was performed with BD FACS Cytofix/Cytoperm and BD
anti-mouse antibodies (CD3-PercP-Cy5.5, CD8-APC-Cy7, CD44-APC, IL-2FITC, IFNg -Pecy7, TNFa-PE, BD Biosciences). Dendritic cell staining was
performed on cell suspensions obtained from the auricular draining lymph
nodes using BD antibodies (CD8a-APC-Cy7, CD11c-PeCy7, CD80-APC,
CD86-PE, MHCII-FITC, BD Biosciences and Clec9A-PE, Miltenyi Biotech
(Macquarie, NSW, Australia)), cells from lymph nodes were first counted using
Trypan blue staining to determine whether they had equivalent cellularity. Cells
were analysed on a BD FACS Canto using the gating strategy described in the
relevant Supplementary Figures and results analysed with FlowJo software.

EcoHIV challenge
EcoHIV/NL4-3 challenge and qRTPCR monitoring were performed as
described previously.5759 Briefly, EcoHIV stocks were prepared by
transfection of pEcoHIV into HEK293T cells. Purified virus supernatant (or
conditioned media control) was administered to mice via intraperitoneal
injection at a dose of 1.5 mg p24 (Zeptometrix p24 antigen ELISA). At 7 days
post EcoHIV challenge, mice were culled and spleen, PECs and peripheral
blood samples were collected. RNA was isolated using the Trizol method and
used to generate complementary DNA (Qiagen Quantitect RTPCR kit). Then,
50 ng of complementary DNA was used as template for quantitative real-time
PCR using the Quantifast Sybr Green kit (Qiagen) to determine levels of MLV
RNA (50 -GAGGTCGGGTGGAAGTACCA-30 and 50 -TGCATCTTGGCCTTT
TCCTT-30 ). Results were normalised to RPL13a mRNA levels (50 -TAGG
GCCAAACCCCGTTCTG-30 and 50 -GCCGGTGGAAGTTGGGTAGG-30 ) after
validation of primer amplification efficiency, using the DDCT method of
quantification.

Statistical analysis
Data are presented as means.e.m. Data analysis and generation of graphs was
performed using Graphpad Prism 5.0b (La Jolla, CA, USA) and SAS Version
9.3 (Cary, NC, USA), with assistance from the Data Analysis and Management
Centre, University of Adelaide. Nonparametric KruskalWallis test was used to
compare the difference between the multiple vaccine groups (pVAX GAG
Standard Vaccine, pVAX GAG DTa (A), pVAX GAG PRF (B) and pVAX GAG
2A PRF(C)). If the global test showed significant difference between the
groups, then Wilcoxon tests were performed to compare the post hoc difference
between Standard Vaccine group vs A, Standard Vaccine group vs B and
Standard Vaccine group vs C separately.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

ACKNOWLEDGEMENTS
We thank Chris Burrell, Ilia Voskoboinik. Simon Barry, Ehud Hauben and Don
Anson for advice, materials and technical support. DTa was subcloned from a
construct originally designed by Hugh Trahair. This work was supported by
Grant Numbers 543143 and APP1026293 from the National Health and
Medical Research Council (NHMRC) of Australia and by Grant Number
BF040005 from the Australia-India Biotechnology Fund. AS and EJG are
Research Fellows supported by NHMRC.

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