|
http://www.abbs.info e-mail:[email protected] ISSN
0582-9879
ACTA BIOCHIMICA et
BIOPHYSICA SINICA 2003, 35(8): 695–701
CN 31-1300/Q |
Antigen-expressed
Recombinant Salmonella typhimurium Driven by an in Vivo-activated
Promoter is Capable of Inducing Cellular Immune Response in Transgenic Mice
WANG
Hong-Wei, ZHANG Min, LUAN Jie, HU Wei-Jiang, ZHAO Ping, GAO Jun, QI Zhong-Tian*
( Department of Microbiology, Second
Military Medical University, Shanghai 200433, China )
Abstract To explore the approaches and mechanisms for reversing the immune
tolerance in transgenic mouse, and the pathogenicity of hepatitis G virus
(HGV), the promoter of phoP-activated gene (PpagC) of Salmonella typhimurium
was used as a transcriptionally regulating element to construct an attenuated
S. typhimurium expressing HGV NS3. The recombinant S. typhimurium was orally
administered to HGV transgenic mice. As the results, HGV antigen in serum and
liver as well as HGV mRNA in liver were decreased significantly, although the
serum anti-HGV NS3 remained undetectable as the control transgenic mice. The spleen
cell proliferation, in vitro HGV NS3 specific CTL, and IFN-γ assays with the primed cultured
splenocytes indicated the induction of Th1 immune responses in those
administered transgenic mice. Adoptive transfer of fractionated primed spleen
cells to the transgenic mice showed that T lymphocytes were responsible for,
maybe through IFN-γ, the down-regulation of HGV
mRNA transcription. Histological examination found no significant inflammatory
changes in liver of the transgenic mice. These findings suggested that the oral
inoculation of the HGV NS3-expressed attenuated S. typhimurium driven by an in
vivo-activated promoter should be a simple and effective approach for potential
treatment of chronic viral infection.
Key
words hepatitis G virus;
transgenic mouse; PpagC; attenuated Salmonella typhimurium ; immune tolerance;
adoptive transfer
Transgenic mouse is a useful system for
studying viruses[1－4]. The transgenic mouse
carrying the entire genomic cDNA of HGV was established previously in our
laboratory[5]. HGV antigens were expressed in various tissues such as liver,
kidney and lung, HGV proteins and RNA were detectable persistently in serum of
the transgenic mice, but there were no cellular and humoral immune responses or
significant pathological changes, which suggested an immune tolerance to HGV.
So it is necessary to break the tolerance of the transgenic mouse for further
investigation on the immunopathology and pathogenicity of HGV. It has been
confirmed that oral immunization is effective to induce cellular and humoral
immune responses, and the vaccines using attenuated Salmonella typhimurium as a
carrier are capable of inducing protective immune reactions in transgenic mice.
Therefore it is possible to develop a dual function vaccine for prevention and
treatment of viral infections[6－8]. However, in the
recombinant attenuated S. typhimurium the exogenous plasmids are easy to lose,
and the persistent-expressed proteins may be toxic to the host. The recently
established expression system, using the promoter of phoP-activated gene
(PpagC) as a transcriptionally regulating element controlled by the phoP/PhoQ
regulator in S. typhimurium[9－11], has been confirmed
helpful in resolving the above problems[12,13]. By changing the culture
conditions of S. typhimurium as increasing Mg2+, Ca2+ or reducing nutrition
components, the activity of PpagC can be restrained[14,15], which lowers the
expression of exogenous gene in recombinant bacteria before inoculation into
the host, and avoids the influences on the recombinant bacteria and the lose of
the exogenous plasmids. When the recombinant bacteria entered into the
macrophages of host, the exogenous gene was initiated promptly by PpagC, the
expressed proteins were processed and presented and induced cellular and humoral
immune responses in host. HGV NS3-expressed recombinant attenuated S.
typhimurium driven by PpagC has been constructed in our lab. When orally
inoculated C57 mice, the recombinant S. typhimurium induced strong cellular and
humoral immune responses[16].
In this study, the recombinant attenuated
S. typhimurium was used to orally administer HGV transgenic mice to further
explore its potential immunotherapeutic effect. Additionally, the pathological
changes in the transgenic mice were observed, and the mechanisms of breaking
the immune tolerance and acquiring immunotherapeutic effects were also
discussed.
1 Materials
and Methods
1.1 HGV transgenic mice
The founder transgenic mice carrying the
complete cDNA were established in our laboratory, and fostered in school’s
transgenic animals center which accords with the SPF criterion. These mice were
allowed to produce offsprings which were screened by PCR and Southern blotting.
HGV antigens expressed in serum and tissues of transgenic mice were detected by
ELISA and immunohistochemical staining. The transgenic mice with higher levels
of antigens were used to produce offsprings.
1.2 Experimental materials
HGV cDNA containing plasmid pHGV18-10, HGV
NS3 protein, B16 cells capable of stably expressing the GFP-HGV NS3 fusion
protein, were prepared previously in our laboratory[16－18]. Attenuated S. typhimurium SL7207
were gifts from Dr. Bruce Stocker (Stanford University, USA). Mouse anti-HGV E2
monoclonal antibody (IgG)[19] was a gift from Dr. Alfred M. Engel (Boehringer
Mannheim GmbH, R&D Infectious Diseases, Nonnenwaldstr, Germany). The
prokaryotic expression plasmid pQE was a product of Qiagen. Mouse IFN-γ and IL-4 detection kits were purchased
from Jingmei Biotech. Non-radioactivity cell proliferation and cell cytotoxic
detection kits were products of Promega. High fidelity Pfu DNA polymerase,
restriction enzymes and T4 DNA ligase were purchased from Sangon Company.
HRP-conjugated goat anti-mouse IgG, G418 and mitomycin C were products of
Sigma. Dig high prime DNA labeling and detection starter kit I was purchased
from Roche Biochemical Company. Female, 6-week old C57 mice were purchased from
the Experimental Animal Center of Xiper-Bikai Company in Shanghai.
1.3 Construction of recombinant plasmid driven by PpagC
The sequence of pagC promoter was
amplified from attenuated S. typhimurium, and used to replace the prokaryotic
promoter of pQE vector. The resulted plasmid was named pZW. HGV ns3 and lacZ gene
fragments were inserted into pZW separately to form pZW-ns3 and pZW-lacZ,
respectively. Then pZW-ns3 or pZW-lacZ was transferred into attenuated S.
typhimurium SL7207 to form SL7207/pZW-ns3 or SL7207/pZW-lacZ(control
bacteria)respectively. SL7207/pZW-ns3 and SL7207/pZW-lacZ were cultured in LB
medium containing different concentrations of Mg2+ to find the most
suitable[Mg2+] restraining the activity of PpagC[16].
1.4 Immunization by oral administration of recombinant attenuated S. typhimurium
Bacteria were amplified according to the
reported method[16]. HGV transgenic mice (n=10) were immunized for three times
at a 3-week interval via oral gavage with 100 μL
PBS containing 107 recombinant attenuated S. typhimurium. Before immunization,
each mouse was fasted from water and food for 4 h, and then received 100 μL 5% NaHCO3 for neutralizing the stomach
acid.
1.5 Adoptive transfer experiments
Primed splenocyte suspensions of
immunized C57 mice were prepared for adoptive transfer using lymphocyte
separating buffer. After four washes with RPMI 1640 medium, lymphocytes were
counted and resuspended in 200 μL PBS. Approximately (5－10)×107
cells were injected into the tail vein of each recipient mouse once it was
sub-lethally irradiated (5 Gy). T and B lymphocytes were further separated by
nylon wool adherence with purities to 95% as assessed by flow cytometry.
Approximately 5×106 T or B lymphocytes from a
single spleen were injected into the tail vein of each recipient mouse that had
been sub-lethally irradiated.
1.6 Serological tests
Serum ALT of the mice was measured by
Auto-Biochemical Analysis Instrument. Serum anti-HGV NS3 was detected according
to the reference[16]. Serum HGV E2 were detected as follows. Each microtiter
well was coated by anti-HGV E2 monoclonal antibodies [1∶5000 dilution with 0.01 mol/L carbonate
buffer (pH 9.6)], which was blocked by the same buffer containing 0.5% BSA. 50 μL sample dilution buffer and serum were
added to per well, and incubated for 45 min at 37 ℃. 100 μL
human anti-HGV IgG was further applied per well and incubated for 45 min at 37 ℃, followed by incubation with 100 μL HRP-conjugated goat anti-human antibody
(1∶1000 dilution) for 30 min at
37 ℃. At last, the wells were
visualized with o-phenylenediamine (OPD) and absorbance at 492 nm was detected.
1.7 Immunohistochemical staining
The livers of mice were fixed in 10%
neutral buffered formalin, embedded in paraffin, and sectioned at a thickness
of 4 μm. The slices were washed with
PBS, and blocked with normal goat serum. Then they were first administrated
with 15 mg/L mouse anti-HGV E2 monoclonal antibodies for 60 min at room
temperature. After being washed with PBS, they were incubated with the
secondary goat anti-mouse IgG (1∶100 dilution) for 30 min at
room temperature. Finally 3,3′-diaminobenzidine (DAB)/H2O2
was used to stain them. A control group was manipulated under identical
conditions.
1.8 Northern blot analysis
Total RNA of mouse liver was extracted by
the routine procedures. 50 μg RNA was fractionated on a 1%
formaldehyde agarose gel and blotted onto nylon membranes. Then the membranes
were hybridized with Dig-labeled DNA probes of HGV NS3 region or β-actin DNA fragments using the random
primed DNA labeling system (Roche) according to the instruction manual.
1.9 Detection of cellular immune response
1.9.1 Tlymphocyte
proliferation assay Spleno-cyte
suspensions prepared in sterile condition were cultured in 96-well plates at 5×105 cells/mL in 200 μL RPMI 1640 medium containing HGV NS3
protein (10 mg/L), BSA (10 mg/L), ConA (5 mg/L), with medium alone as a
negative control. Each group was set up 3 wells. After 72 h, each group was
treated with MTS for 1 h, then A590 values were detected and the stimulating
index (SI) was calculated according to the formula(1), in which n was
sequenced number of antigen stimulating group sample and m was sequenced number
of negative control group.
(1)
1.9.2 Detection
of CTL response B16
cells stably expressed GFP-HGV NS3 fusion protein served as target cells, and
the primed splenocytes from immunized mice as effector cells. The CTL responses
were detected by lactic dehydrogenase-release assay, and the results were shown
as the killing ratio of effector cells to target cells as previously
described[20].
1.10 Cytokine assays
Splenocyte suspensions were prepared as
above and adjusted to 7×106 cells/mL. Add 100 μL cell suspensions to each well of
96-well plates, and set up non-stimulating group, ConA stimulating group, BSA
stimulating group and HGV NS3 stimulating group. Culture supernatants were
collected after 48 h of incubation. The concentrations of IFN-γ and IL-4 were determined by ELISA using
commercial kits.
1.11 Histological analysis
The livers of mice were obtained by
operation and sliced. The HE staining was performed by routine pathological
procedure and the pathological changes were observed through optical
microscope.
1.12 Statistical analysis
Data in this experiment were analyzed by
Student’s t-test.
2 Results
2.1 Influence on expression of serum HGV E2 in transgenic mice after immunization or adoptive transfer
Anti-HGV NS3 antibodies remained
undetectable in serum of transgenic mice at the 2nd, 5th, 8th, and 11th week
after first inoculation. Serum ALT levels of both immunized and non-immunized
transgenic mice were in normal scope (data not shown). HGV E2 proteins were
detectable in the serum of the immunized group. The inoculation of SL7207/pZW-ns3
into HGV transgenic mice induced a gradual decrease of serum HGV E2. At the
11th week after first immunization, HGV E2 became undetectable. Compared with
the immunized mice, HGV E2 didn’t significantly decrease in control
mice[Fig.1(A)]. Serum HGV E2 of transgenic mice was also measured at 5th, 10th,
15th, 20th and 25th day post-transfer of primed splenocytes, T or B lymphocytes
of littermate normal mice immunized with the recombinant bacteria. The results
showed that the circulating HGV E2 decreased in the serum of transgenic mice
adoptive transferred primed splenocytes and T lymphocytes but not in B
lymphocytes group[Fig.1(B)].
Fig.1 Influence on concentration of
serum HGV E2 in transgenic mice after immunization or adoptive transfer
experiments
The concentrations of serum HGV E2 in
transgenic mice are indicated with the absorbance at 492 nm (A492). (A)
Concentration of serum HGV E2 in transgenic mice at weeks 0, 2, 5, 8, 11 after
immunization with SL7207/pZW-ns3(□)or
SL7207/pZW-lacZ(○).
(B) Concentration of serum HGV E2 in transgenic mice at days 0, 5, 10, 15, 20,
25 after adoptive transfer of primed splenocytes (●)、
T lymphocytes(■)
or B lymphocytes(▲).
2.2 Cellular immune responses
There were no significant proliferation
of splenocytes in both experimental and control group stimulated with BSA. But
there were significant proliferations in splenocytes of experimental group
(immunized with SL7207/pZW-ns3) stimulated with HGV NS3 compared with control
group immunized with SL7207/pZW-lacZ (P<0.05, Table 1). Cyto-toxicity
was measured using splenocytes from immunized mice as effector cells (E) and
B16 (cells stably expressed GFP-HGV NS3 fusion protein) astarget cells (T). The
results showed that the killing ratios increased significantly in experimental
groups at different E ∶T ratios (20∶1 or 100∶1)
compared with the corresponding control groups (P=0.001 and 0.0003,
respectively, Table 2).
|
Table 1 Proliferation of T lymphocytes
from immunized HGV transgenic mice
SI,
the stimulating index. |
Table 2 HGV NS3 specific CTL responses of
immunized HGV transgenic mice
E:T,
effector cells vs. target cells. |
2.3 Influence on the expression of HGV E2 protein in the liver tissues of transgenic mice after immunization or adoptive transfer
The immunohisto-chemical staining was
conducted in livers of the mice 2 weeks after the third inoculation with the
recombinant bacteria or the mice 25 d after adoptive transfer of primed cells.
The results indicated that HGV E2 expression decreased significantly in the
livers of SL7207/pZW-ns3 immunized mice and mice transferred with primed
splenocytes or T lymphocytes (Fig.2). However, there was no significant
influence on HGV E2 expression in the livers of mice adoptive transferred with
primed B lymphocytes (data not shown).
Fig.2 Influence of immunization or
adoptive transfer on the expression of HGV E2 protein in the liver tissues of
transgenic mice(200×)
(A) Transgenic mouse immunized with
SL7207/pZW-lacZ. (B) Transgenic mouse immunized with SL7207/pZW-ns3. (C)
Transgenic mouse adoptively transferred with primed spleen cells. (D)
Transgenic mouse adoptively transferred with primed T lymphocytes.
2.4 Influence of immunization or adoptive transfer on the transcription of HGV mRNA in the livers of transgenic mice
The contents of HGV mRNA in the livers of
immunized transgenic mice were analyzed by Northern blotting. The results
indicated that HGV mRNA could be detected in the livers of normal transgenic
mice and transferred transgenic mice with primed B cells. However, the HGV mRNA
were undetectable in the livers of transgenic mice after 3 times immunization
with the recombinant bacteria, or those 15 d after transferred with primed
splenocytes or T lymphocytes (Fig.3).
Fig.3 Influence of immunization or
adoptive transfer on the transcription of HGV mRNA in the livers of transgenic
mice
Dig-labeled DNA probes specific for HGV
mRNA and β-actin
were used. Northern blot analysis was carried out on 50 μg
of total RNA isolated from the livers of immunized transgenic mouse. 1,
SL7207/pZW-ns3; 2, adoptively transferred with primed T lymphocytes; 3,
adoptively transferred with primed B lymphocytes; 4, SL7207/pZW-lacZ.
2.5 Influence on cytokines secretion of splenocytes from transgenic mice inoculated with recombinant bacteria
Splenocytes suspensions from mice at the
11th week after first immunization were obtained and cultured in vitro. ELISA
was performed to measure the cytokines secreted by the cultured splenocytes.
The results showed that IFN-γ was detectable, however, IL-4
was undetectable in both normal and transgenic mice (Table 3).
2.6 Influence on histology of the livers
of transgenic mice orally inoculated with recombinant bacteria
Table 3 Detection of cytokines in spleen cells from mice
immunized with SL7207/pZW-ns3
|
Mice |
Non-Tg |
Tg |
||
|
Cytokines |
IL-4 |
IFN-γ |
IL-4 |
IFN-γ |
|
Medium |
3±2 |
5±3 |
2±1 |
3±1 |
|
ConA |
59±13 |
1308±299 |
36±11 |
903±187 |
|
HGV NS3 |
4±2 |
289±99 |
6±3 |
199±68 |
Splenocytes from SL7207/pZW-ns3 immunized
mice were incubated with medium or stimulated with concanavalin A (ConA, 2.5 g/L)
or HGV NS3 (102.5 mg/L) for 48 h. The culture supernatants were harvested for
determination of cytokine levels (ng/L). The data were the x±s
of 3 spleens. Non-Tg, non-transgenic mice; Tg, transgenic mice.
Routine pathological analysis showed that
there were only mild changes such as lymphocyte invasion in the livers of
transgenic mice after 3 times immunizations with the recombinant bacteria or
transgenic mice 15 d after transfer with primed splenocytes or T cells. Fig.4
indicated the histology in the livers of controlled transgenic mice and
transgenic mice orally immunized with recombinant bacteria.
Fig.4 Histological analysis of the
livers of transgenic mice (100×)
(A)
HE staining of the liver of transgenic mouse immunized with SL7207/pZW-lacZ.
(B) HE staining of the liver of transgenic mouse immunized with
SL7207/pZW-ns3.3Discussion
Using HGV full-length genomic cDNA clone (GenBank
No: AF081782), we established HGV transgenic mice[5]. The transgenic mice are
tolerant to HGV, although HGV proteins express and virus RNAs replicate. There
were no significant histological changes in the transgenic mice. The small
transgenic animals of HGV carrier provided a perfect experimental model for
exploring the approaches to reverse the immune tolerant state in chronic virus
hepatitis. We once attempted to break the tolerance in the transgenic mice by
muscle injection of the expression plasmids containing genomic HGV cDNA or by
oral inoculation with the attenuated S. typhimurium constructed through
traditional method, but no expected results occurred (data not shown).
In this present study, we amplified PpagC
sequence from S. typhimurium and constructed a novel target antigen expression
system driven by the in vivo-activated promoter. This system was introduced
into attenuated S. typhimurium and demonstrated to help enhance stability of
the transferred plasmids as well as induce stronger humoral and cellular immune
responses than traditional methods in mice[21]. HGV NS3-expressed recombinant
attenuated S. typhimurium driven by the in vivo-activated pagC promoter also
induced strong humoral and cellular immune responses in normal C57 mice[16].
HGV NS3 protein participates in HGV
polyprotein processing and virus RNA replication, and is a key protein in the
life cycle of HGV[22－24]. In addition, HGV ns3 gene
is highly conservative. So recombinant bacteria SL7207/pZW-ns3 was construct to
orally immunize HGV transgenic mice, and HGV NS3 would be expressed as a target
antigen. The results showed that specific cellular immune response was induced
in transgenic mice although anti-HGV NS3 antibodies were undetectable in the
serum of immunized mice. Interestingly, concentrations of serum HGV antigens,
expression of HGV antigens and contents of HGV mRNA in the livers decreased
significantly. These findings indicated that the immune tolerance to HGV
antigens was broken by oral immunization, which resulted in an inhibition of
the HGV replication and expression (including HGV E2). HGV mRNA, namely HGV
genomic RNA, was undetectable in the livers of immunized transgenic mice by
Northern blotting, which was consistent with the results in protein level.
Adoptive transfer experiments demonstrated that T cells were responsible for
the down-regulation of HGV replication and expression. The cultured spleen
cells from immunized normal and transgenic mice secreted IFN-γ but not IL-4 when stimulated with HGV
NS3, which indicated the induction of Th1 immune response in transgenic mice,
and suggested that HGV replication and expression were down-regulated probably
by IFN-γ. IFN-γ affects different aspects of the
specific immune response through up-regulating the expression of class I and
class II MHC molecules[25]. In general, HGV antigens are higher in the livers
than in other tissues of transgenic mice, and almost undetectable in the thymus
of adult mice (data not shown). Additionally, they also maintained at
relatively low levels in serum. However HGV antigens were highly expressed by
recombinant bacteria in host antigen presenting macrophages with a higher
quantity in comparison with that expressed by transgenic mice themselves, which
might be the key mechanism for the break of tolerance.
HGV replication and expression were down
regulated effectively through the oral immunization, which benefited the immune
capacity. Interestingly this HGV NS3-specific immune response didn’t cause
histological damages in the livers or increase serum transaminase activity. The
absence of a detectable cytotoxic attack on the HGV NS3 expressing liver cells,
even after transfer of fully competent T cells, may be due to poor expression
of MHC class I molecules on hepatocytes[26].
It was recently reported that when
co-infected with HGV, HIV replication level decreased in HIV infected persons,
clinical symptoms were relatively mild and survival period prolonged in AIDS
patients[27,28]. On the other hand, the attenuated S. typhimurium is proved
safe as a carrier of recombinant oral vaccines, and the commercial Ty21a is the
human attenuated Salmonella. So it is necessary to study HGV and its
interaction with host cells, which might provide a competent approach for treating
chronic viral infection through oral immunization with recombinant Ty21a using
PpagC to drive antigens.
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_________________________________________
Received:
April 15, 2003 Accepted: May 20, 2003
This
work was supported by the grants from the National High Technology Research and
Development Project of China (863 Program) (No. 2002AA214161) and the National
Natural Science Foundation of China (No. 30170514)
*Corresponding
author: Tel/Fax, 86-21-25070312; e-mail, [email protected]