Further analysis of protection induced by the MIC3 DNA vaccine against T. gondii: CD4 and CD8 T cells are the major effectors of the MIC3 DNA vaccine-induced protection, both Lectin-like and EGF-like domains of MIC3 conferred protection.
ABSTRACT The present study was conducted mainly to evaluate the contribution of the cellular and the humoral responses in protection conferred by the MIC3 DNA vaccine (pMIC3i) that was proved as a potent vaccine against toxoplasmosis. We performed the adoptive transfer of CD4(+) and CD8(+) T lymphocytes from pMIC3i immunized mice to naive ones and the role of humoral immunity was evaluated by in vitro invasion assays. We also constructed plasmids encoding the EGF-like domains and the Lectin-like domain of MIC3, to define which domains of MIC3 are involved in the protection. Furthermore, the adjuvant effect of the GM-CSF-expressing vector (granulocyte-macrophage colony-stimulating factor) required the precise temporal and spatial codelivery of GM-CSF with antigen, thus, we constructed a bicistronic plasmid expressing MIC3 and GM-CSF. In conclusion, the protection induced by pMIC3i was mainly mediated by CD4(+) and CD8(+) T lymphocytes and both EGF and Lectin domains of MIC3 conferred protection. Furthermore, the codelivery of GM-CSF by a bicistronic plasmid appeared to be a most effective way for enhancing the adjuvant properties of GM-CSF.
- SourceAvailable from: Carlos Henryque Souza e Silva[Show abstract] [Hide abstract]
ABSTRACT: BACKGROUND:: The aim of this study was to evaluate the association between clinical signs of congenital toxoplasmosis and IgG subclasses found in newborns participating in the Minas Gerais State Neonatal Screening Program. METHODS:: Neonates with confirmed congenital toxoplasmosis underwent standardized ophthalmologic evaluation, neuroimaging studies and hearing assessment, as well as ELISA testing for total IgG (IgGt) and its subclasses (IgG1, IgG2, IgG3 and IgG4) against soluble (STAg) and recombinant (rSAG1 and rMIC3) antigens of Toxoplasma gondii. RESULTS:: Newborns with congenital toxoplasmosis but without ocular lesions were more likely to present anti-rMIC3 IgGt when compared with those newborns with active or cicatricial retinochoroidal lesions. Detection of anti-rMIC3 IgG2 and IgG4 was associated with presence of retinochoroidal lesions and intracranial calcifications, with higher mean reactivity index (RI) values than unaffected newborns with congenital toxoplasmosis. Anti-STAg IgG3 was associated with newborns without neurologic damage. CONCLUSIONS:: Specific subclasses of IgG antibodies reacting with recombinant antigens of T. gondii may serve as biomarkers of neurological and ocular changes in newborns with congenital toxoplasmosis.The Pediatric Infectious Disease Journal 08/2012; · 3.57 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Toxoplasmosis caused by the protozoan Toxoplasma gondii is a major public health problem, infecting one-third of the world human beings, and leads to abortion in domestic animals. A vaccine strategy would be an ideal tool for improving disease control. Many efforts have been made to develop vaccines against T. gondii to reduce oocyst shedding in cats and tissue cyst formation in mammals over the last 20 years, but only a live-attenuated vaccine based on the S48 strain has been licensed for veterinary use. Here, the authors review the recent development of T. gondii vaccines in cats, food-producing animals and mice, and present its future perspectives. However, a single or only a few antigen candidates revealed by various experimental studies are limited by only eliciting partial protective immunity against T. gondii. Future studies of T. gondii vaccines should include as many CTL epitopes as the live attenuated vaccines.Expert Review of Vaccines 10/2013; · 4.22 Impact Factor
- 03/2012; , ISBN: 978-953-51-0274-8
Vaccine 27 (2009) 2959–2966
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/vaccine
Further analysis of protection induced by the MIC3 DNA vaccine against T. gondii:
CD4 and CD8 T cells are the major effectors of the MIC3 DNA vaccine-induced
protection, both Lectin-like and EGF-like domains of MIC3 conferred protection
Alaa Bassuny Ismaela, Dorsaf Hedhlia, Odile Cérèdeb, Maryse Lebrunb,
Isabelle Dimier-Poissona, Marie-Noëlle Mévéleca,∗
aUniversité Franc ¸ois Rabelais, INRA, UMR 0483 Université-INRA d’Immunologie Parasitaire, Vaccinologie et Biothérapies anti-infectieuses,
IFR 136 Agents transmissibles et Infectiologie, UFR des Sciences Pharmaceutiques, 31 avenue Monge, 37200 Tours, France
bUniversité de Montpellier, CNRS, UMR 5539 Université-CNRS, Université de Montpellier 2, 34090 Montpellier, France
a r t i c l ei n f o
Received 12 January 2009
Received in revised form 27 February 2009
Accepted 28 February 2009
Available online 13 March 2009
MIC3 DNA vaccine
CD4 and CD8
a b s t r a c t
The present study was conducted mainly to evaluate the contribution of the cellular and the humoral
responses in protection conferred by the MIC3 DNA vaccine (pMIC3i) that was proved as a potent vaccine
We also constructed plasmids encoding the EGF-like domains and the Lectin-like domain of MIC3, to
define which domains of MIC3 are involved in the protection. Furthermore, the adjuvant effect of the GM-
and spatial codelivery of GM-CSF with antigen, thus, we constructed a bicistronic plasmid expressing
MIC3 and GM-CSF. In conclusion, the protection induced by pMIC3i was mainly mediated by CD4+and
CD8+T lymphocytes and both EGF and Lectin domains of MIC3 conferred protection. Furthermore, the
codelivery of GM-CSF by a bicistronic plasmid appeared to be a most effective way for enhancing the
adjuvant properties of GM-CSF.
© 2009 Elsevier Ltd. All rights reserved.
Toxoplasma gondii, is the causative agent of toxoplasmosis, a
life-threatening disease in immunocompromised patients and a
potentially severe disease in immunosuppressed patients and con-
genitally infected children [1,2]. In animals, toxoplasmosis is of
great economic importance worldwide due to abortions, stillbirth
and neonatal losses, especially in sheep and goats .
Primary infection with T. gondii results in the development of
both humoral and cell-mediated immune responses and confers
long-term protection. This suggests that the development of an
effective vaccine is a realistic goal.
Protection against T. gondii infection is mainly attributed to
cell-mediated immunity (for reviews see refs. [4,5]). Specific T lym-
phocytes act either as cytokine producer cells that help infected
cells to kill the parasite or as cytotoxic cells that destroy infected
cells. Previous studies have shown that both CD4+and CD8+T cell
subtypes are involved in the protection [6–12].
∗Corresponding author. Tel.: +33 2 47 36 71 86; fax: +33 2 47 36 72 52.
E-mail address: firstname.lastname@example.org (M.-N. Mévélec).
Infection with T. gondii also stimulates humoral immunity, both
against T. gondii remained unclear until the use of B-cell-deficient
mice by Kang et al.  and Johnson and Sayles . Their results
indicated the importance of the B-cell Ab response in preventing
persistent proliferation of tachyzoites in the brain and lung during
the chronic phase of infection.
In molecular terms, invasion of host cells by T. gondii is closely
coupled to the release of proteins stored within apical secretory
granules known as micronemes, which play a central role in the
recognition of and adhesion to host cells (for reviews see refs.
[17,18]). Among these proteins, the protein MIC3 contains adhe-
sive motifs and has been shown to bind to the surface of host
cells [19,20]. MIC3 contains five epidermal growth factor-like (EGF)
domains and the Lectin-like domain required for binding . The
domain IV and part of the EGF-like domain V, was shown to con-
tain human B and T cell epitopes that are recognized by adults with
acquired T. gondii infection and children with congenital infection
We have demonstrated that MIC3 of T. gondii is a potent
and effective vaccine candidate against toxoplasmosis. Thus, the
plasmid DNA encoding the immature form of the MIC3 protein
0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.
A.B. Ismael et al. / Vaccine 27 (2009) 2959–2966
combined with pGM-CSF, a plasmid encoding the granulocyte-
macrophage colony-stimulating factor, induced strong specific
humoral and cellular immune responses, as well as highly effec-
tive significant protection against the chronic phase of infection in
In the present study, we evaluated the contribution of the
cellular and humoral anti-MIC3 immune responses to protection
induced by intramuscular (i.m.) immunization of CBA/J mice with
fer of CD4+and CD8+T lymphocytes from pMIC3i immunized
mice to naive ones and the role of humoral immunity was assayed
by in vitro invasion assay. In addition, we have constructed a
plasmid encoding the EGF-like domains (pcDNA3.1 EGF “pEGF”)
and a plasmid encoding the Lectin-like domain (pcDNA3.1 Lectin
“pLectin”) of MIC3 in order to define which domains are involved
in the protection. Furthermore, since the adjuvant effect of the
GM-CSF-expressing vector required the precise temporal and spa-
tial codelivery of GM-CSF with antigen [24–26], we constructed a
bicistronic plasmid expressing both MIC3 and GM-CSF.
2. Materials and methods
Six-to eight-week-old female CBA/J mice (H-2k) were obtained
from Janvier, Le Genest St Isle, France, and maintained under
pathogen-free conditions in our animal house for use throughout
these experiments. Experiments were performed in accordance to
the rules of the Ethic Committee from Tours University.
RH strain tachyzoites were harvested from the peritoneal fluids of
Swiss OF1 mice that had been intraperitoneally infected 3–4 days
earlier. The 76K strain cysts were obtained from the brains of orally
infected CBA/J mice and maintained by monthly passage.
2.3. Preparation of T. gondii antigen (TAg)
The T. gondii RH tachyzoites were washed, sonicated and cen-
trifuged as previously described . The protein concentration
was determined in the supernatant (later used as the source of
antigen) by the Micro BCA protein assay reagent kit using bovine
serum albumin (BSA) as standard (Pierce, Rockford, USA). The TAg
was stored at −80◦C until use.
2.4. Adoptive transfer of CD4+and CD8+T lymphocytes
In adoptive transfer experiments, spleens were aseptically
removed at week 6 from nine mice immunized with either empty
plasmid (pcDNA3) or pMIC3i combined with pGM-CSF as previ-
ously described . Briefly, mice were injected using syringes
with 30.5-gauge needles with 100?l of cardiotoxin (10?M): solu-
tions were injected into each tibialis anterior muscle 5 days before
DNA immunization, to enhance the uptake of the plasmid DNA
. On days 0 and 14, the regenerating muscles of these mice
were injected with 50?g pcDNA3 or with 50?g pMIC3i combined
with 50?g pGM-CSF, in 100?l sterile endotoxin-free PBS (50?l
in each muscle) and then boosted on day 28 with 50?g of plas-
mid pcDNA3 or pMIC3. The spleens were pooled and single-cell
viously described . A positive selection for either CD4+or CD8+
splenocytes from each group of mice was performed with a MACS
LS+separation column (Miltenyi Biotec, Bergisch Gladbach, Ger-
many) according to the manufacturer’s instructions. We evaluated
the purity of the two subpopulations by flow cytometric analysis
(Becton Dickinson; FACScan cytometer) using CellQuest software
and monoclonal antibodies, anti-CD4 (L3T4) and anti-CD8a (LY2)
(Pharmingen), respectively. We obtained a purity of >80% for each
subpopulation after LS-MACS separation. Naive mice were injected
intravenously (i.v.) via the tail vein with 7×106cells in 0.2ml of
phosphate buffered saline (4 groups of 6 mice). Twenty-four hours
after cell transfer, the mice were orally challenged with 60 cysts of
the 76K strain. The mice were killed 1 month later.
2.5. In vitro invasion assay
The in vitro invasion assay was performed as previously
described . Briefly, 2×104BHK-21 cells were seeded in 96-well
plates (Costar) in 200?l of culture medium (GIBCO BRL) sup-
plemented with 5% fetal calf serum (FCS), 2% tryptose, 100U/ml
penicillin and 100?g/ml streptomycin. Plates were incubated for
24h at 37◦C in 5% CO2, 95% humidity. The tachyzoites were incu-
bated for 30min at room temperature with 1:10 dilution of sera
of mice immunized with pMIC3i combined with pGM-CSF or sera
of mice immunized with pcDNA3 alone, in cell culture medium.
A serum from a T. gondii-infected mouse was included as a posi-
tive control of inhibition. The immune and control sera-tachyzoites
mixtures were then added to each corresponding well at a 5:1, par-
(specific activity 50Ci/mmol) were added to each well followed by
incubation for 16h. Host cells and Toxoplasma were lysed by freeze
thawing, harvested by aspiration, and were collected on fiberglass
results are expressed as mean cpm (counts per minute)±SD.
2.6. Plasmid constructions
The primers used for constructions are listed in Table 1.
Plasmid pMIC3i has been constructed previously to express
the complete MIC3-ORF (RH(ERP) strain, GenBank accession no.
AJ132530) . Briefly, the complete MIC3-ORF (open reading
frame) was obtained by PCR amplification of MIC3 from pBluescript
ment was inserted into the eukaryotic expression vector pcDNA3
Plasmid pEGF was designed to express the EGF-like domains
of MIC3. The signal sequence and the EGF-like domains of MIC3
were PCR-amplified independently with primers ML5/ML6 and
ML3/ML4, respectively. The two XbaI-digested PCR fragments were
ligated together. The ligation product was PCR-amplified with
primers ML5/ML4. The PCR fragment was inserted into the NheI
and HindIII sites of pcDNA3.1 (Invitrogen). Plasmid pLectin was
designed to express the Lectin-like domain of MIC3. The signal
sequence and the Lectin-like domain of MIC3 were PCR-amplified
independently with primers ML5/ML6 and ML1/ML2, respectively.
ation product was PCR-amplified with primers ML5/ML2. The PCR
fragment was inserted into the NheI and HindIII sites of pcDNA3.1
Primers used for DNA constructs.
A.B. Ismael et al. / Vaccine 27 (2009) 2959–2966
(Invitrogen). The bicistronic plasmid pIRES-MIC3i-GMCSF was
designed to coexpress MIC3i and GM-CSF from the same plasmid.
The pIRES plasmid (Clontech) allows the simultaneous expres-
sion of two genes by cloning them into two separate multiple
cloning sites (MCSA and MCSB) located on either side of the inter-
nal ribosome entry site (IRES) from the encephalomyocarditis virus
(EMCV). Both MCS and the IRES sequence are downstream from the
from the second MCS, SV40 polyadenylation signals direct the cor-
rect processing of the 3?end of mRNA. The GM-CSF sequence was
obtained from the pGM-CSF plasmid by BamHI and XhoI digestions
and treatment with Klenow. The fragment was inserted into the
SmaI site of the MCSB of pIRES. The MIC3i sequence was gener-
ated by PCR from plasmid pMIC3i with primers 5?MIC3i-IRES and
3?MIC3i-IRES. PCR products were digested with NheI and MluI and
inserted into the MCSA of pIRES. pIRES utilizes a partially disabled
second downstream cloned gene. To augment the rate of transla-
the SmaI site (at position 1745) on the pIRES vector were deleted.
This modification generated pIRESopt with a single SmaI site in the
MCSB. The GM-CSF and MIC3i sequences were inserted into pIRE-
The plasmid encoding murine granulocyte-macrophage colony-
stimulating factor (pGM-CSF) was kindly provided by H.C.J. Ertl
(Wistar Institute, Philadelphia, PA).
2.7. Assay for in vitro GM-CSF expression
GM-CSF expression by monocistronic and bicistronic vectors
was tested by transiently transfecting 293 FT cells using a polyca-
by the manufacturer. One day after the transfection, the culture
supernatants of transfected 293 FT cells were assayed to determine
2.8. Plasmid purification
All the plasmids were purified from transformed E. coli DH5? by
anion-exchange chromatography (EndoFree plasmid Giga or Mega
kit, Qiagen GmBH, Hilden, Germany), following the instructions
provided by the manufacturer. The purified plasmids were dis-
solved in sterile endotoxin-free phosphate-buffered saline (PBS;
Sigma) and stored at −20◦C. The integrity of the DNA plasmids
was checked by agarose gel electrophoresis after digestion with the
mined by absorbance at 260nm. The OD 260/280 ratios for purified
DNA were 1.80–1.95, indicating that preparations were free of any
major protein contamination.
2.9. DNA immunization
The mice (10–11 per group) were immunized intramuscularly
syringes with 30.5-gauge needles (Microlance, Becton Dickinson),
with 100?l of 10?M cardiotoxin (Latoxan, Rosans, France); the
solutions were injected into each tibialis anterior muscle 5 days
before DNA immunization to enhance the uptake of the plasmid
DNA . On days 0 and 14, the regenerating muscles of these mice
50?g pGM-CSF, in 100?l sterile endotoxin-free PBS (50?l in each
muscle) and then boosted on day 28 with 50?g of plasmid alone.
The control group was composed of mice injected i.m. with 50?g
of the empty plasmid, pcDNA3, three times at intervals of 2 weeks.
2.10. Enzyme-linked immunosorbent assay (ELISA)
Levels of antigen-specific IgG antibodies in serum samples were
determined as previously described . The TAg at 10?g/ml was
used to coat microtiter plates. The antigen-specific antibody titer
is given as the reciprocal of the highest dilution producing an
absorbance (OD) that was 2.5-fold greater than that of the serum of
mice injected with the empty plasmid, pcDNA3, at the same dilu-
tion. Results are expressed as the means of log2titers±standard
2.11. In vitro lymphocyte proliferation studies
viously described . Splenocytes (three mice per group) were
stimulated with various concentrations of TAg between 0.5 and
15?g/ml. The incorporated radioactivity (cpm) was measured by
liquid scintillation counting. The results are expressed as ?cpm,
calculated as the cpm obtained in the presence of the stimulating
antigen minus the cpm in the medium alone.
2.12. Cytokine assays
Spleen cell proliferation was assayed as previously described
, the cells were stimulated by incubating with 15?g/ml of
TAg or with the medium alone. Cell-free supernatants were har-
vested and assayed for interleukin-2 (IL-2) and interleukin-4 (IL-4)
activities at 24h, interleukin-10 (IL-10) activity at 72h and for
gamma-interferon (IFN-?) activity at 96h. The concentrations of
IL-2, IL-4, IL-10 and IFN-? were determined by ELISA kit (OptEIATM
Set, Pharmingen, San Diego, USA), following the instructions pro-
2.13. Challenge infection
CBA/J mice were infected orally with 60 cysts of the 76K strain
two weeks after the last immunization. One month after the chal-
lenge, mice were killed and their brains were removed. Each brain
The mean number of cysts per brain was determined microscopi-
cally by counting eight samples (10?l each) of each homogenate.
2.14. Statistical analysis
Levels of significance of the differences between groups of mice
were determined by Mann–Whitney U test or two-tailed Student’s
3.1. Both CD4+and CD8+T lymphocytes from mice vaccinated
with pMIC3i combined with pGM-CSF are involved in protection
We previously shown that mice immunized with pMIC3i dis-
played significant protection against an oral challenge with 76K
T. gondii strain cysts, exhibiting fewer brain cysts than the con-
trol mice immunized with the empty plasmid pcDNA3. The
co-administration of pGM-CSF enhanced this protection. The
ability of plasmid GM-CSF to augment the immunogenicity of
DNA vaccines required spatial codelivery of antigen and GM-CSF.
Because of preliminary studies demonstrating that GM-CSF by
itself (i.e., without the antigen MIC3) did not affect the parasite
burden compared to mice immunized with empty pcDNA3, con-
trol mice used in this study were immunized with pcDNA3 alone
A.B. Ismael et al. / Vaccine 27 (2009) 2959–2966
Fig. 1. Adoptive transfer of CD4+and CD8+T lymphocytes. CD4+and CD8+T lymphocytes were enriched from mice immunized with pMIC3i combined with pGM-CSF or from
mice immunized with the empty plasmid pcDNA3 (control) and injected by i.v. injection into four groups of naive mice (n=6). After 24h, the mice were orally challenged with
60 cysts of T. gondii strain 76K. The brain cyst load was determined 1 month after the challenge. The solid bars indicate the median. P<0.002 versus control (very significant),
(pcDNA3: 5468±1048 cysts/brain; pcDNA3 mixed with pGM-CSF:
5187±1622 cysts/brain; n=9).
Splenocytes from CBA/J mice vaccinated i.m. with pMIC3i com-
bined with pGM-CSF or empty plasmid pcDNA3 were enriched for
CD4+and CD8+T lymphocytes. The enriched subpopulations were
after 24h with 60 cysts of the 76K strain. CBA/J mice immu-
nized with 7×106CD4+or CD8+T lymphocytes from pMIC3 plus
formation 1 month after oral challenge than those mice receiving
T lymphocytes provide significant protection against oral T. gondii
infection (62% and 54% brain cyst reduction, respectively).
3.2. MIC3-immune serum did not inhibit infection of cultured
cells by T. gondii tachyzoites
Replication of anti-MIC3-pretreated tachyzoites in BHK-21 was
mice immunized with pMIC3i combined with pGM-CSF did not
inhibit tachyzoite replication (Fig. 2). Thus, anti-MIC3 antibodies
may not function protectively in vivo to block infection of host cells
3.3. The EGF-like domains of MIC3 are the major inducers of a
To determine the levels of antibody titers in immunized mice,
all sera were tested by ELISA using TAg (Fig. 3). A high level of
specific anti-MIC3 IgG antibody titers was found in all sera of
by T. gondii within BHK-21 cells. Tachyzoites were incubated for 30min in cell cul-
ture medium with serum from pMIC3i-immunized mice, pcDNA3-immunized mice
or serum from an infected mouse at 10% of the final concentration. The mixtures
were then added to BHK-21 cells for 2h, and then pulsed with [3H]-uracil for 16h.
Toxoplasma proliferation was determined by beta counter. Values shown are the
mean cpm±SD of the triplicate samples. The data are representative of two sepa-
rate experiments performed. RH alone refers to BHK-21 cells cultured with T. gondii
incubated only with media. Cell alone refers to cpm from uninfected BHK-21 cells.
Serum of infection refers to a serum from a T. gondii-infected mouse, used as a posi-
tive control of inhibition. (*) Significant inhibition compared to RH alone (P<0.005,
mice immunized with pEGF combined with pGM-CSF, and these
titers were increased with the booster. Anti-MIC3 IgG antibod-
ies were detected in 50% of the immunized mice with pLectin
combined with pGM-CSF and were detected only after the last
booster (third immunization). By contrast, mice injected with the
control plasmid pcDNA3 generated no antibodies against MIC3
3.4. EGF and Lectin domains of MIC3 are inducers of cellular
To evaluate cellular anti-Toxoplasma immune responses in the
MIC3 DNA-vaccinated CBA/J mice, splenocytes from mice immu-
nized with pMIC3i, pEGF or pLectin, combined with pGM-CSF,
were prepared 2 weeks after the third immunization and were
stimulated in vitro with TAg (Fig. 4). Marked highly specific lym-
phoproliferation was observed in splenocyte cultures from mice
immunized with pMIC3i combined with pGM-CSF (?cpm, 8000
at 15?g/ml of TAg). A slight but specific dose-dependent lym-
phoproliferation was observed in splenocyte cultures from mice
immunized with pEGF or pLectin combined with pGM-CSF (?cpm,
3000 at 15?g/ml of TAg). In contrast, no specific lymphoprolifer-
ation was observed in splenocyte cultures from mice immunized
with the control plasmid pcDNA3 when stimulated with TAg. In
addition, splenocytes from all immunized and control groups pro-
liferated to comparable levels in response to the mitogen, Con A
(IS>10, data not shown). Therefore, both EGF and Lectin domains
of MIC3 contain T epitopes.
The supernatants of splenocytes cultured from mice immu-
nized with pMIC3i, pEGF or pLectin, combined with pGM-CSF
were harvested at various times after the restimulation with
TAg, and were assessed for the production of IL-2, IFN-?, IL-4
and IL-10 cytokines. Specific amounts of IL-2 were synthesized
by the restimulated splenocytes from the mice immunized with
Fig. 3. Determination of specific anti-MIC3 antibody titers in the sera of CBA/J mice
immunized with 50?g pMIC3i, pEGF or pLectin combined with 50?g GM-CSF-
encoding plasmid on days 0 and 14, and then boosted on day 28 with 50?g of
each plasmid. Sera were collected on days 14, 21 and 35 and tested by ELISA using
T. gondii antigens. The titer is given as the reciprocal of the highest dilution with an
absorbance that was 2.5-fold greater than the absorbance of untreated mice sera at
the same dilution. Results are expressed as the mean log2titers±SD and are repre-
sentative of one of two experiments. (*) Anti-MIC3 antibodies were detected in 50%
of mice immunized with pLectin combined with pGM-CSF.
A.B. Ismael et al. / Vaccine 27 (2009) 2959–2966
Fig. 4. In vitro proliferation and cytokine production of splenocytes from CBA/J mice (3 mice per group) immunized with 50?g pMIC3i, pEGF or pLectin combined with 50?g
GM-CSF-encoding plasmid on days 0 and 14, and then boosted on day 28 with 50?g of every plasmid, in response to T. gondii antigen. Control splenocytes were isolated from
mice receiving the empty plasmid, pcDNA3. The results of proliferation are expressed as ?cpm, calculated by subtracting the mean counts per min of unstimulated cells from
the mean counts per min of stimulated cells. Cell-free supernatants from stimulated splenocytes (TAg, 15?g/ml) were harvested and assayed for IL-2 at 24h and for IFN-?
activity at 96h. Results of cytokines are representative of one of two experiments.
pMIC3i, pEGF or pLectin combined with pGM-CSF (Fig. 4). Signifi-
cantly large amounts of IFN-? were produced in the supernatants
of restimulated splenocyte cultures from mice immunized with
pMIC3i (59.6±0.015ng/ml) combined with pGM-CSF (Fig. 4). Spe-
cific amounts of IFN-? were also produced in the supernatants
of restimulated splenocyte cultures from mice immunized with
pEGF (18.87±7.74ng/ml) or pLectin (9.5±2.46ng/ml) combined
with pGM-CSF, but to a less degree than that produced by spleno-
cytes from pMIC3i-immunized mice (Fig. 4). By contrast, no
specific release of IL-4 and IL-10 from any culture supernatant was
demonstrated (data not shown). These results indicate that a Th1
immune response was induced by immunization with MIC3 DNA
and both EGF and Lectin domains of MIC3 are inducers of this
3.5. Both EGF and Lectin domains of MIC3 are involved in
To test whether vaccination with plasmid DNA encoding EGF
or Lectin domains of MIC3 induced effective protection against
T. gondii infection, the immunized mice were orally challenged 2
weeks after the last immunization with 60 cysts of T. gondii 76K
strain. One month after the oral challenge, the cysts in the brains
of the mice were counted (Fig. 5). Effective and highly significant
protection was displayed in mice immunized with pEGF, pLectin
or pMIC3i, combined with pGM-CSF (P<0.001 vs mice immunized
brain cysts than mice immunized with pcDNA3 (negative control)
nized with 50?g pMIC3i, pEGF or pLectin combined with 50?g GM-CSF-encoding
plasmid on days 0 and 14, and then boosted on day 28 with 50?g of each plasmid.
The vaccinated mice (n=7) were orally challenged 2 weeks after the last immuniza-
after challenge. Mice inoculated with empty plasmid pcDNA3 were used as control.
The solid bars indicate the median. Data shown are representative of one of two
individual experiments. P<0.001 with respect to control (Mann–Whitney test).
(Fig. 5). Thus, both EGF and Lectin domains of MIC3 are involved in
protection against T. gondii infection. Similar results were obtained
without pGM-CSF codelivery (data not shown).
3.6. The codelivery of GM-CSF by a bicistronic plasmid provides
the most effective procedure for enhancing the adjuvant
properties of GM-CSF
To further improve the effects of GM-CSF, we adopted a strategy
based on a bicistronic vector. Bicistronic plasmids use an internal
the ribosome to attach to mRNA and translate the downstream
coding sequence, while the upstream sequence is translated by
expression, internal initiation mediated by viral IRESs is often
lower . For this reason, we chose to insert the GM-CSF gene
in the second cistron of pIRES. Furthermore, EMCV IRES sequence
insertion into pIRES mutated AUG 11. Initiation in type II IRES
usually occurs at the AUG 11 codon. The initiation codon AUG 11
is located downstream from a sequence of about 20 nucleotides
(a spacer designed Xm), preceded by a poly-pyrimidine tract (Yn
tract; n=5–7 nucleotides). The length of the Yn tract and the Xm
spacer is important for IRES function. In pIRES, the AUG that is
used for secondary translation is contained in the sequence of
the inserted gene. The distance between the Yn tract and the
AUG is as such increased and, therefore, a less efficient transla-
tion of the downstream gene is observed (Clontech). Indeed, the
amount of GM-CSF produced in the culture supernatant of cells
transfected with pIRES-MIC3i-GMCSF (Xm=43) was 26-fold lower
than that produced by monocistronic expression (12.6ng/ml vs
327ng/ml). To increase the translational efficacy, the length of Xm
was reduced (Xm=22 nucleotides). As expected, the amount of
GM-CSF produced in the culture supernatant of cells transfected
with pIRESopt-MIC3i-GMCS was higher than that transfected with
pIRES-MIC3i-GMCSF; however, this was still lower (about 8-fold
The ability of mammalian cells to secrete MIC3i after transfec-
pcDNA3-MIC3i was tested by Western blotting after probing with
anti-MIC3i mice sera. MIC3i expression levels in cells transfected
with pIRESopt-MIC3i-GMCS and in cells with the monocistronic
vector were similar (data not shown).
The efficacy of the bicistronic DNA vaccine was compared with
that of the monocistronic pMIC3i vector mixed with pGMCSF
(Table 2). Groups of CBA/J mice were immunized with 10?g of
pIRESopt-MIC3i-GMCSF three times at 2-week intervals or with
A.B. Ismael et al. / Vaccine 27 (2009) 2959–2966
The protective efficacy of MIC3 was improved by the use of a bicistronic vector.
GroupBrain cysts numbera(n=8)IgG titersalog2(n=11) Cytokine productionaIFN-? (ng/ml) (n=3)
4077 ± 1860
1796 ± 528b
3480 ± 803
– 0.71 ± 0.4
3.12 ± 0.83d
2.44 ± 1.2
10 ± 0.0c
9 ± 1.5
aValues are means±SD.
bP<0.0001 (Student’s t test), extremely significant.
cAnti-MIC3 antibodies were detected in three mice.
dP=0.010 (Student’s t test), significant compared to untreated mice.
(See Section 2). Control mice were left untreated. After the chal-
pGMCSF and control mice. However, mice vaccinated with 10?g of
pIRESopt-MIC3i-GMCSF had 56% fewer brain cysts than the control
Compared with controls, spleen cells from pIRES-vaccinated
mice produced particular levels of IFN-? if stimulated with TAg
(3.12±0.83ng/ml vs 0.71±0.4ng/ml). This was not the case in
spleen cells from pcDNA3-vaccinated mice (2.44±1.2ng/ml vs
vaccinated with pIRES generated antibodies against MIC3.
We have previously shown that a DNA vaccine encoding the
MIC3 protein of T. gondii elicits strong specific cellular and humoral
immune responses, as well as providing an effective protection
against T. gondii infection in CBA/J mice. Also, a combination of a
plasmid encoding GM-CSF enhanced the immune response and the
protection provided .
Here, we first investigated the contribution of the cellular and
humoral anti-MIC3 immune responses to protection following the
coinjection of two plasmids, one encoding MIC3 and the other
GM-CSF. DNA vaccination is a technique with strong potential to
elicit cell-mediated immunity, triggering antigen-specific produc-
tion of IFN-? and priming CTL responses . Multiple studies
have demonstrated the ability of plasmid GM-CSF to augment the
immunogenicity of DNA vaccines. These reports include increased
immunization regimens, and specific assays used. In our experi-
mental conditions, we did not know if GM-CSF promote the levels
of CD4+or CD8+or both.
IFN-? [8,34,35], CD4+and CD8+T cells are the main players
involved in resistance to T. gondii, during both acute and chronic
phases of infection [6,9,36]. In agreement with those findings, our
results showed that both CD4+and CD8+T cells from pMIC3i-
immunized mice are involved in protection against the chronic
phase of infection.
The relative contributions of CD4+and CD8+T cells primed
by DNA vaccines against T. gondii infection were also investigated
by Nielsen et al.  and Scorza et al.  by adoptive transfer
or in vivo depletion. In adoptive transfer experiments, naive mice
that received CD8+T cells from SAG1 DNA-vaccinated mice had
extended survival times after challenge with RH strain . In vivo
depletion of CD8+T cells eliminated the protective effect of the
GRA1 DNA vaccine against acute toxoplasmosis, whereas depletion
of CD4+T cells had no influence on mortality . However, in vivo
CD4+T cell depletion led to an increase in brain cyst burden dur-
ing the chronic phase of infection. These previous experiments and
our findings here provide no conclusive information on the DNA
vaccine-elicited effector mechanism: direct CD8+T cell cytolysis or
Indeed, both CD8 cytolytic activity and IFN-? secretion can con-
Perforin-deficient mice vaccinated with ts4 resist infection by the
highly virulent RH strain, suggesting that control of acute infec-
tion does not require CTL activity. However, CTL function appears
to play a role in host protection during the later stages of infec-
tions, with perforin-deficient mice being slightly more susceptible
tion, ?2-microglobulin-deficient mice, lacking functional CD8+T
cells could survive acute T. gondii infection; however, they become
more susceptible to the chronic phase . CTL function is possi-
bly involved in the prevention of cyst reactivation or, alternatively,
may limit the number of parasites initially encysting within tis-
sues. However, tachyzoites within a cell targeted for cell lysis are
not killed by the cytolytic event . CTL activity may spread infec-
tion by release of tachyzoites. Alternatively, tachyzoites released by
CTL lysis may then be susceptible to phagocytosis and inactivation
by cells as suggested by Denkers .
CD4+T cells are important for early IFN-? production during T.
gondii infection and an absence of CD4+leads to parasite multipli-
cation in the tissues . CD4-deficient mice did exhibit increased
parasite burdens in the brain . CD4+T cells, through production
of IL-2, are important for the induction of CD8+T cell response in T.
gondii-infected hosts, as well as for the maintenance of CD8+T cell
effector immunity . CD4+T cells also contributed significantly
to protection against chronic infection via their role as helper cells
for production of isotype-switched antibodies .
To elucidate whether B-cells or antibodies play a protective role
in resistance against infection with T. gondii, B-cell-deficient mice
(?MT) have been used. These mice have normal Ag-presenting
function for priming CD4+T cells, as well as unimpaired CD8+
T cell response. T. gondii-infected ?MT mice survived the acute
phase of the infection but died 3–4 weeks after infection. In these
mice, mortality was associated with continuous proliferation of
tachyzoites. Expression of IFN-?, TNF-? and inducible NO syn-
thase did not differ between infected ?MT mice and controls .
Adoptive transfer of anti-T. gondii IgG Abs to infected ?MT mice
zoites in the brain . In addition, the resistance to a challenge
infection with virulent RH tachyzoites can be markedly improved
in ts4-immunized ?MT mice by administration of serum from
donors immunized with T. gondii . Antibodies may function
protectively in vivo by blocking infection of host cells by tachy-
zoites rather than functioning as part of FC receptor-dependent
or complement-dependent anti-Toxoplasma effector mechanism
It was shown from studies cited above that antibodies may play
a role against T. gondii infection. Thus, we evaluated in vitro the
effect of anti-MIC3 immune sera on the infection of cells by the
RH strain of T. gondii. Pretreatment of tachyzoites with anti-MIC3
suggesting that anti-MIC3 Abs do not inhibit T. gondii cell invasion.
Micronemes are apical secretory organelles, presumed to play a
A.B. Ismael et al. / Vaccine 27 (2009) 2959–2966
predominant role in the early phase of the invasion process by dis-
charging adhesins capable of interacting with host cell receptors
of the host cells and the surface of the parasites . The receptor
binding site of MIC3 is located in the N-terminal chitin binding-
like domain. The N-terminal pro-peptide cleavage and C-terminal
dimer formation are needed to allow the expression of the binding
property . pMIC3i expressed a dimeric recombinant MIC3 that
the pro-peptide, which is not cleaved by mammalian cells and does
not bind to the cell surface . Thus, with the recombinant MIC3,
conformation”. Garcia-Réguet et al.  have previously shown
that rabbit polyclonal antibodies against immunoaffinity-purified
MIC3 and mouse infection sera interfere with cell binding to MIC3.
However, these antibodies did not interfere with invasion . In
addition, the anti-MIC3 4.2F3 monoclonal antibody, of which the
binding site is located within EGF domain of MIC3, has no effect on
invasion . Together, previous results and those reported here
suggest that either the antibody is unable to reach the target in the
organisms, or it does not have the necessary affinity and/or speci-
ficity. Whether T. gondii infection, immunoaffinity-purified MIC3
or pMIC3i immunization induced antibodies directed against the
alternative pathways can substitute MIC3 in invasion. Indeed, dele-
tion of MIC3 generated mutants that were still able to invade cells
. However, immunization of B-cell-deficient mice with pMIC3i
could definitively demonstrate whether B-cells may or may not be
required for resistance to T. gondii.
by pMIC3i is closely related to CD4+and CD8+T lymphocytes. It
was of interest, to know the MIC3 domains likely to express major
T epitopes involved in protection. Therefore, we constructed plas-
mids encoding the EGF-like domains (pEGF) and the Lectin-like
domain (pLectin) of MIC3. All mice immunized intramuscularly
with pEGF combined with pGM-CSF and 50% of mice immunized
with pLectin combined with pGM-CSF developed a specific anti-
MIC3 humoral response as determined by ELISA using TAg, which
contained the mature form of MIC3. These findings suggested that
B epitopes are located within the EGF domains. Alternatively, mice
struct used in this study is similar to the construct used by Cérède
et al. . These authors previously showed that the Lectin-like
A slightly specific lymphoproliferation was observed in spleno-
with pGM-CSF when stimulated with TAg. This cellular immune
response was associated with IFN-? and IL-2 synthesis. By contrast,
no specific release of IL-4 and IL-10 from any culture supernatant
was shown. These findings indicated that a Th1 immune response
was induced and that both EGF and Lectin domains of MIC3 are
inducers of this response.
To test whether vaccination with plasmid DNA encoding EGF
or Lectin domains of MIC3 contributed to the protection against T.
tive and highly significant protection was obtained in the mice
immunized with pEGF, pLectin or pMIC3i, combined with pGM-
CSF: these mice, respectively, had 56%, 60% and 70% fewer brain
cysts than mice immunized with pcDNA3 (negative control). Thus,
both EGF and Lectin domains of MIC3 are involved in protection
against T. gondii infection, suggesting that both domains contain
major T epitopes involved in protection. Further studies are needed
to define the protective T cell epitopes. Potential T cell epitopes
could be predicted by computer algorithms.
To further enhance the protective efficacy of MIC3, the entire
CSF was introduced into the bicistronic vector, pIRES. To optimize
the expression of GM-CSF, the pIRES vector was modified (pIRE-
Sopt). However, the expression of GM-CSF was still below that of
MIC3-GMCSF had fewer cysts than control mice. Also, splenocytes
from these mice produced more IFN-? than mice immunized with
10?g of pMIC3i plus pGM-CSF, not protected against brain cyst for-
mation. These results are consistent with previous data showing
that the effect of GM-CSF was increased by a bicistronic DNA vac-
cine strategy [26,45,46]. The use of one vector instead of two, at
least at low doses, is important for optimal protection against T.
gondii, within the context of cocktail DNA vaccines against T. gondii.
In conclusion, the protection induced by the DNA MIC3 vac-
cine was mainly mediated by cellular immunity. Furthermore, both
EGF and Lectin domains of MIC3 are involved in the protection. A
bicistronic vector designated to express both MIC3i and GM-CSF
improved the protective efficacy of the MIC3 gene.
This work was supported by grants from the Conseil Général
d’Indre et Loire and from the Région Centre.
We appreciate the gift of GM-CSF plasmid provided by H.C.J.
Ertl. We thank, T. Fandeur, Thi Cam Tu Le, J. Pierre, J.M. Rith, and T.
Papin for technical assistance. We also thank S. Bigot for secretarial
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