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Introduction
Hepatitis B virus (HBV) infection remains a serious worldwide health
problem[1]. Approximately 5%_15% of infected
adults and 90%_95% of infected newborns fail to clear infection and become chronic carriers who would face greater risk of
developing cirrhosis and hepatocellular carcinoma later in
life[2]. Although prophylactic vaccination using the recombinant
HBV surface antigen was introduced more than 20 years ago and has been a great success, existing carriers still account for
up to 20% of the population in certain Asian and African
countries[3]. Therefore, there is an urgent need for new and effective
therapeutic approaches to treat these patients. The success of prophylactic vaccine brought high hopes towards the
development of immunotherapy approaches against
existing viral infection to complement the current interferon
treatment and antiviral chemotherapy. Agents such as the
hepatitis B (HB) subunit vaccine, cytotoxic T lymphocyte (CTL)
epitope vaccine, Hepatitis B vaccine and anti-HBs complex
all have been under investigation for such
purposes[4_6].
Among all the approaches, DNA-based vaccination
agents seem most promising for their ability to elicit both
strong humoral and cellular
immunities[7,8]. Further formulation and delivery methods are being developed to augment
the immunogenicity, such as using viral
vectors[9],
micros-pheres[10], gene
gun[11], or electroporation
(EP)[12,13]. We adopted the EP-mediated delivery approach and showed that
the EP-mediated HBV surface antigen (HBsAg) encoding
DNA vaccination resulted in much higher antibody titers
and stronger CTL responses in healthy animals and
non-human primates[14].
To evaluate the therapeutic potential of such a
vaccination approach, it would be more important to demonstrate
viral clearance resulting from the treatment. Several earlier
studies used a transgenic mouse model containing the HBV
genome as the relevant disease
model[15]. However, since the HBV genes were expressed at low levels throughout the
animal and not limited in the liver tissues as in a real human
infection, transgenic mice are not considered suitable for
evaluating antiviral drugs or therapeutic methods, especially
when observing the clearance process histologically in the
liver tissues. In this study, we developed a model system in
which liver cells were transfected and made to express a
large quantity of HBsAg using the hydrodynamic DNA
injection method to test the clearance ability of immune
response induced by EP-mediated HBV DNA
vaccination[16,17]. We showed that not only the secreted viral proteins, but
also those in the cytoplasm of hepatocytes, were quickly
eliminated, without apparent cytotoxicity in EP-mediated
vaccination animals.
Materials and methods
Plasmid and animals Plasmid DNA encoding the HBV
surface antigen preS2-S[18] was constructed using the
pcDNA3.1 vector as described previously and named
pHBS[19]. Recombinant hepatitis B vaccine (rec-HBs) was purchased
from Kangtai Biological Products (Shenzhen, China). Healthy
female BALB/c mice, 6_8 weeks old, were purchased from
Fudan University Animal Facility (Shanghai, China), where
they were housed in compliance with the regulations of the
China Council for Animal Care. Their use in this experiment
and all the experimental procedures conformed to the
guideline of the Shanghai Experimental Animals Management.
Animal immunization The mice were anaesthetized and
shaved to expose the injection site above the gastrocnemius
muscle on 1 hind leg; then were administered the injections
of 5 µg pHBS or 0.5 µg rec-HBs. For the EP-mediated DNA
vaccination (pHBS/EP), the mice were treated with EP within
1 min after the injection using the following parameters: 60
V/cm field strength, 6 pulses, and 50 ms duration with a 1 s
interval between pulses.
Hydrodynamic transfection 10 µg HBV surface antigen
expression plasmid in sterile saline in a volume equal to 10%
of mice body weight was rapidly injected into tail vein.
Measurement of HBsAg level and anti-HBs titers
Blood samples were collected at specific time points and sera were
isolated for analysis. For the muscle and liver samples, the
mice were killed at designated time points and the whole
liver or entire muscle fiber was removed and homogenized in
cell lysis solution. The amount of HBsAg and the HBV
surface antibody (anti-HBs) titers was measured using the
corresponding ELISA kits (Sino-American Biotechnology,
Shanghai, China). The HBsAg standards in µg/mL and
anti-HBs standards in mIU/mL were also purchased from the
Sino-American Biotechnology Company. The antigen level and
antibody titers were calculated based on the standard curve
obtained.
ELISpot assays The cellular immune responses of the
mice after vaccination were tested with an ELISpot assay
using the IFN-g ELISpot kit (R&D Systems, Minneapolis,
MN, USA). Spleen cells were isolated 2 weeks after
vaccination and washed 3 times in RPMI medium, then plated into
the blocked ELISpot plate at the density of
1×106 cells per well. The HBsAg mouse epitope peptide with the sequence
of LTKILTIPQSLDSWWTSLN was synthesized by Shenergy
Biocolor Bioscience and Technology (Shanghai, China) for
antigen-specific stimulation. The ELISpot assay was carried
out according to the manufacturer's protocol. The blue spots
in each well were counted manually under a dissection
microscope.
Immunohistochemical assay of HBsAg expression in the
liver At a specific time after the hydrodynamic injection, the
mice were killed and the liver portions were immediately fixed
in 10% buffered formaldehyde, embedded in paraffin,
sectioned at 5 µm thickness, and mounted on slides. For the
HBsAg immunohistochemical assay, tissue sections were
incubated for 10 min with 3% (v/v)
H2O2 and blocked with 5%
(w/v) bovine serum albumin in PBS for 10 m at 20_25
oC. Goat anti-HBs (dilution 1:1000; Dako, Carpinteria, CA, USA) was
then added and kept incubated for 1.5 h at room temperature.
After being washed, the sections were incubated with the
biotinylated secondary antibody, followed by a
streptavidin-horseradish peroxidase complex in accordance with the
manufacturer's protocol (LSAB staining kit, Dako, USA).
Finally the reactions were visualized in a solution containing
3, 3'-diaminobenzidine tetrahydrochloride and Meyer's
hematoxylin counterstaining. The surface antigen-positive
cells were counted from 4 different views of the liver
sections from 6 different animals in each group.
Histological examination of the liver tissues
The tissue sections were prepared according the method mentioned
above, and the histological changes were examined by HE
staining.
Alanine aminotransferase assay Serum alanine
aminotransferase (ALT) levels were measured at various time
points using an alanine aminotransferase kit (Fosun
Long March Medical Science, Shanghai, China) and an
ANALYTECH-738PLUS biochemical analyzer (ANTAI Diagnostics, Shanghai, China).
Statistical analysis Unless otherwise stated, the results
are presented as mean±SD. Statistical analyses were made
using Student's t-test.
Results
Improved immune responses resulting from
EP-mediated DNA vaccination The EP treatment greatly enhanced
the transfection efficiency of the DNA vaccine in the
gastrocnemius muscles of the mice (Figure 1A). With a 5
µg intramuscular-injected pHBS dose, the HBsAg expressed in
muscles was approximately 2.0 ng at 24 h after injection; with
the EP-mediated delivery, approximately 30 ng HBsAg was
obtained. The antibody titer in the EP group reached 300
mIU/mL 4 weeks after the vaccination, and remained at high
levels (>1000 mIU/mL) for at least 6 months after only 1
immunization (Figure 1B). Cell-mediated immunity was also
examined using antigen-specific T cell ELISpot assays.
Splenocytes from mice immunized with pHBS/EP responded
more readily to HBs epitope stimulation (Figure 1C),
compared to the mice immunized by pHBS without EP or treated
with EP only.
Reduction of HBsAg expression in the liver in
immunized mice We used the hydrodynamic injection method to
forcefully and effectively transfect the liver cells of the mice
to mimic an existing infection model. The hydrodynamic
injection method had been widely used to deliver plasmid
DNA into hepatocytes by quickly injecting a large volume of
solution in a few seconds, in which the gene transfection is
based on physical forces only and is unrelated to the
vaccination status of the animals. In naive mice, the
hydrodynamic injection of 10 µg pHBS resulted in considerable
HBsAg expression in the liver within 12 h after the injection,
and lasted for about 72 h, but in pHBS/EP-immunized mice
with the same dose of pHBS, the HBsAg level in the liver
lysates was much lower at all the detection time points. In
comparison, the change of HBsAg in the mice injected with
the pHBS without EP was similar to naive mice (Figure 2A).
We also compared the HBsAg levels in rec-HBs-vaccinated
mice and in pHBS/EP-vaccinated mice (Figure 2B). At
approximately 4 weeks after vaccination, both groups had
high antibody titers. A hydrodynamic injection of pHBS
resulted in high circulating HBsAg in the serum in naive
mice within 12 h, but in both vaccinated groups, serum HBsAg
was almost undetectable due to the neutralization effect of
the antibodies. In the liver lysates, the situation was
different; the amount of HBsAg in the pHBS/EP-vaccinated
group was much lower than that in the protein-vaccinated
group.
Immunohistological analysis of HBsAg expression in
immunized mice After the hydrodynamic injection of pHBS
in the mice, the expressed HBsAg in the liver sections was
examined by immunohistochemical staining for the HBV
surface antigen (Figure 3). In the naive mice, after
hydrodynamic injection, HBsAg-positive hepatocytes were easily
visible with brown staining throughout the hepatic lobule
within 12 h after the injection. Both cytoplasmic and nuclear
staining was present. The staining was the heaviest around
d 1 after the hydrodynamic injection, and gradually faded
from d 2 to 7. This is consistent with the liver HBsAg ELISA
measurements described earlier. In comparison, in the
pHBS/EP vaccinated mice, the HBsAg-positive hepatocytes were
significantly fewer (Table 1) and the color of the stained cells
was much lighter.
Histological analysis of the liver tissues in immunized
mice The histological changes in the liver tissues after the
hydrodynamic treatment and during the viral antigen
clearance process were examined by HE staining of the liver
sections (Figure 4). In naive mice, the hydrodynamic injection
procedure did not seem to have caused any lasting tissue
damage to the liver. Even in the pHBS/EP-vaccinated
animals, the liver cells looked normal. Notably, there was no
visible hepatocyte toxicity, tissue inflammation, or even
lymphocyte infiltration observed.
Change of ALT The change of the ALT value was also
measured to probe liver tissue toxicity with the
hydrodynamic treatment and during the antigen clearance process.
The data measured at various time points are listed in
Table 2. There was a transient and rapid increase of the serum ALT
value immediately after the hydrodynamic injection in both
naive and pHBS/EP-vaccinated mice, but they dropped back
to close to the normal range within 48 h. There was basically
no difference between the naive and the
pHBS/EP-vaccinated groups.
Discussion
DNA vaccination has been regarded as one of the most
promising strategies in orchestrating immunological attacks
against viral infections for therapeutic purposes. Its ability
to elicit cell-mediated immune responses is considered
essential for viral clearance and disease eradication. In this
study, we showed that DNA vaccination mediated by EP
resulted in a strong HBsAg specific antibody and
cell-mediated immune responses in mouse models. The results are
consistent with several other studies demonstrating the
immunopotentiating effect of EP-mediated DNA
delivery[12,13].
Specifically for HBV, although recombinant subunit
protein vaccines have been generally successful as a
prophylactic and are used widely, there are more than 350 million
existing virus carriers worldwide who are still waiting for
effective treatment. Studies of the mechanisms underlying
the immunopathogenesis of HBV infection have suggested
that successful resolution of infection was
associated with strong T cell-mediated immune
responses[20,21]. In particular,
CTL that can recognize HBV antigens in hepatocytes and
release antiviral cytokines such as interferon-gamma
(IFN-g) were believed to be critical for viral
elimination[22,23]. In this regard, many studies adopted the DNA-based vaccination
strategy aimed at specifically bringing out cellular-mediated
immune responses. To demonstrate the therapeutic
potential of these approaches for virus elimination,
Oka et al vaccinated the transgenic mice with a high dose of HBV DNA
vaccine encoding the HBV envelope protein, and showed
that 1 single injection of HBV DNA vaccine resulted in the
clearance of serum HBsAg in 28 out of 30 HBV-transgenic
mice[15]. Similarly, Pancholi et al
showed that DNA-based immunization led to the long-lasting inhibition of HBV
replication in a chimpanzee that had been carrying HBV for 12
years. Notably, they reported that the resulting decline of
the covalently-closed circular DNA level coincided with the
rise of IFN-g secreting cell numbers, but not
CTL[24].
To evaluate whether the strong antibody and cellular
responses we observed after EP-mediated DNA vaccination
could help to clear existing viral infections in the liver, we
developed in this study a pseudo-existing infection model
using the hydrodynamic transfection method, which could
transfect as much as 40% of hepatocytes[16,
17]. Because of its liver specificity and high efficiencies, it is usually used to
generate a transient liver-targeted transgenic model for the
study of HBV biology and antiviral
therapy[25], but unfortunately, the duration of such a transfection is rather
short. So in our case, it was impossible for us to vaccinate
after the transfection and build up immune responses. We
had to pre-immunize the mice to establish the full bloom
immune responses before the hydrodynamic transfection, but
the hydrodynamic transfection method delivers genes into
hepatocytes with physical force, by temporally disrupting
the cell membrane and pushing the plasmid DNA in. The
pre-established immune responses should not have any
effect on the transfection process. So from the view of the
immune system, the transfected hepatocytes resembled
existing infection, rather than a new infection.
In DNA/EP-immunized mice, the immune system
immediately mounted a strong immunological attack to hepatocytes
as soon as they started to produce HBsAg. The HBsAg
levels detected in the liver were consistently low, compared to those
in naive mice, rec-HBs-, or DNA-vaccinated mice. During
such a viral antigen clearance process, we observed almost
no histological evidence of cell toxicity, suggesting that the
antigen clearance is largely due to the down regulation of
HBsAg gene expression in liver cells, not cell lysis. This is
similar to what Guidotti et al reported in their study after
injecting HBsAg-specific CTL clones to HBV transgenic
mice[26]. They observed a clearance of HBV mRNA and
serum HBsAg, and found that the downregulation of HBsAg
was not caused by the cytotoxicity effect of CTL, but rather,
by cytokines secreted by the CTL, including IFN-g. However,
in their studies, severe hepatitis was observed, that we did
not see based on the ALT measurement. We think it might
be due to the short duration of viral antigen expression in
transfected hepatocytes by the hydrodynamic injection
method.
We acknowledge that the present study does not
represent a thorough therapeutic model, as during a normal
infection, HBV is able to induce some type of immune
suppression that could make the DNA vaccination strategy less
effective. Further studies in a real, chronically-infected
disease model are necessary in order to understand the
complete process of viral clearance after EP-mediated DNA
vaccination.
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