ABBS 2005,38(06): Immunogenicity of the Plasmodium falciparum Merozoite Surface Protein 1 (C-terminal 19 kD) expressed as a non-glycosylated polypeptide in Pichia pastoris

Abstract        The C-terminal region of the merozoite
surface protein 1 (MSP119)
is one of the most promising vaccine candidates against the erythrocytic forms
of malaria. In the present study, a gene encoding Plasmodium falciparum
MSP119 was expressed in the methyl
hydrate yeast Pichia pastoris. A non-glycosylated form of the
recombinant protein MSP119 was purified from culture medium. This
recombinant protein maintains its antigenicity. Significant immune responses
were seen in C57BL/6 mice after the second immunization. Moreover, the specific
antibodies recognized the native antigens of P. falciparum. The
prevailing isotypes of immunoglobulin (Ig)G associated with immunization were
IgG1, IgG 

Key words        Plasmodium falciparum; merozoite surface protein 1
(MSP119); pichia pastoris; immunity; mice

Malaria
is a major public health problem, resulting in approximately 300-500 million clinical
cases and an estimated 1-3 million deaths each year [1]. Development of a
vaccine against Plasmodium falciparum, the parasite responsible
for the most severe form of malaria, is an urgent priority, particularly
because resistance to most traditional drugs is widespread. Identification of
the targets of naturally acquired protective immunity is an essential component
of vaccine development.

Among
the blood-stage antigens identified, the major surface protein 1 (MSP1) has
been implicated as a target for protective immunity by various criteria in
studies of P. falciparum in nonhuman primates, in vitro studies of
laboratory parasite host models and epidemiological studies of naturally
acquired immunity [2–4]. The C terminus of MSP1, from which MSP119 is
derived, has been studied in most detail as the target of a protective immune
response [5]. The available evidence suggests that antibody against MSP119 can
inhibit parasite growth in vivo and in vitro [6]. Thus, MSP1, especially MSP119
of P. falciparum, is also regarded as a leading vaccine candidate for
malaria.

An
abundant source of recombinant protein, which was produced in a secreted
soluble form with a conformation resembling the native protein, would greatly
facilitate pre-clinical vaccination studies using MSP119. We chose the Pichia
pastoris system not only because it is a eukaryote, which has many
advantages of higher eukaryotic expression systems, such as protein processing,
protein folding, and post-translational modification, but because it is manipulated
as easily as Escherichia coli or Saccharomyces cerevisiae [7]. It is
faster, easier and less expensive to use than other eukaryotic expression
systems, such as Baculovirus or mammalian tissue cultures, and generally gives
higher expression levels. In many cases, it provides a proper folding for
recombinant proteins without the hyperglycosylation in S. cerevisiae [8].

In
the present study, high-level expression of recombinant proteins were obtained
in P. pastoris and most of them were non-glycosylated forms under the
optimized expression condition. Here, we describe some of the immunogenic
properties of recombinant protein MSP119 produced as a secreted
non-glycosylated protein in P. pastoris.

Materials and Methods

Plasmid and bacteria

Expression
plasmid pPIC9k/ MSP119 containing the His 6 tag and coding sequence was
constructed in our laboratory [9]. E. coli DH5a was used as a host for cloning
and P. pastoris GS115 was used for protein expression.

Enzymes, reagents and animals

Restriction
enzymes were purchased from New England Biolabs ( 

Expression of the recombinant
protein MSP119

Transformation
and screening for multiple inserts in GS115 was performed as previously
described [9]. A MutS multiple copy transformant was initially grown overnight
in 5 ml of MGY medium (1.34% YNB, 1% glycerol, 4 mg/ml biotin) in a 100 ml
baffled flask, then was grown at 28–30 ºC in a shaking incubator (300
rpm) for approximately 24 h (OD6006). This 5 ml of culture was inoculated to  

Protein purification and
immunoblotting analysis

MSP119
protein was purified from the fermentation medium using a Ni-nitrilotriacetic
acid (NTA) column. The supernatant was extensively dialyzed against phosphate
buffer at 4 ºC then applied to a Ni-NTA column (Qiagen, Hilden, Germany). The
column was washed three times with washing buffer (pH 8.0; Protein
bands were transferred from the gel to a nitrocellulose membrane using a
Trans-Blot Electrophoretic Transfer Cell (Bio-Rad,  

Animal immunization

The
diluted antigen solution was formulated with CFA or IFA adjuvant at a ratio of
3:7 using a homogenizer at 2500 rpm for 5 min. The quality of the emulsion was
controlled by the droplet test. BALB/c female mice 6 to 8 weeks old and 18 to  

Enzyme-linked immunosorbent
assay (ELISA) for detection of mouse antibody titers to MSP119

Ninety-six-well
plates were coated with 100 ml of native antigen solution (1 mg/ml) diluted in ELISA
for the detection of mouse IgG subclasses was carried out as described above,
except that the secondary antibodies were antibodies specific for mouse IgG1,
IgG 

Indirect immunofluorescent
assay

Thin
blood smears containing the schizont of P. falciparum were prepared, air
dried, and fixed in 100% methanol for 2 min at -20 ºC. The slides were rinsed
in PBS and immersed in blocking buffer (1% bovine serum albumin and 0.1% Triton
X 

Growth inhibition assay

Function
of specific antibodies was determined by detecting their ability to inhibit
growth of the parasite in vitro. Parasites of the FCC1/HN isolate were
maintained in RPMI 1640 medium containing 15% rabbit sera. To isolate specific
antibodies for the inhibition assay, purified recombinant proteins were
immobilized on CNBr-activated Sepharose 4B (Pharmacia Biotech, Uppsala, Sweden)
according to the manufacturer’s instructions. The antibodies eluted from the
columns were extensively dialyzed before use. The inhibition assay was carried
out with the starting culture containing 2% hematocrit and approximately 0.5%
parasitemia with the majority being late trophozoites and schizonts. One
hundred and seventy microliters of the culture suspension and 30 ml of various
concentrations of antibodies were added in triplicate wells to 96-well
flat-bottomed plates and incubated at 37 ºC for 24 h. Thin blood smears were
prepared to determine parasitemia. The inhibition rate was determined according
to the following equation:

where
Pc is the parasitemia of IgG isolated from pre-immune sera and Pt is the
parasitemia of IgG from immune sera.

Cell-mediated immunity

C57BL/6
mice were immunized as described above with recombinant proteins emulsified in
CFA. Twelve to 14 d after immunization, cells were obtained by draining the
lymph nodes of three animals. Cells were washed three times in plain RPMI
medium and re-suspended in 1 ml of cell culture containing RPMI 1640 medium (pH
7.4) supplemented with  

Statistical analysis

Student’s
t test and one-way ANOVA were used to compare the possible differences
in the mean values.

Results

Expression and purification of
MSP119

After
transforming pichia GS115 cells, approximately 300 His+ clones transformed with
the plasmid pPIC9K/ MSP119 were screened for high copy number integration by
G418 selection. Of these clones, six were resistant to 4 mg/ml G418. The
culture supernatant of yeast p. pastoris GS115 transformed with pPIC9K/MSP119
was harvested and concentrated. The target protein was purified by the Ni-NTA
chromatograghy method from the supernatant. To separate the glycosylated and
non-glycosylated forms of MSP119, the purified protein was applied to a
ConA-Sepharose column. Most of the proteins flowed through and the fraction
obtained included non-glycosylated forms. The fractions containing the target
protein were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis
[Fig. 1(A)] and by
immunoblotting using biotinylated ConA [Fig. 1(B)].
There was a small amount of the glycosylated form of MSP 

Evaluation of mouse immune
responses elicited by immunization with MSP119

The immunogenicity
of MSP119 was evaluated after immunization of C57BL/6 mice. As shown in Fig.
2, after the second immunization, the specific antibody levels from
C57BL/6 were significantly increased. Control animals immunized with the
adjuvants only had a negligible antibody immune response to antigen (T=7.648,
P=0.0016).

IgG isotype analysis in mice

As
shown in Fig. 3, four
subclasses of IgG against MSP119 were induced in C57BL/6 mice, IgG1, IgG 

Interaction of specific
antibodies with native antigens on surface of parasite

Fluorescen
merozoites were clearly visible (green) after incubation with the immune sera
and indicated that parasites could be recognized by anti-MSP119 antisera [Fig.
4(C)], but the controls using pre-immune sera had no
fluorescent parasites [Fig. 4(D)],
and parasite nuclei were stained with DAPI [Fig. 4(E,F),
blue]. Antibodies to MSP119 recognized the native antigens of FCC1/HN.

In
vitro growth
inhibition assay

To analyze
the function of antibodies against MSP119, isolated IgG was detected by its
ability to inhibit the invasion of parasites in vitro. As shown in Fig.
5, IgG isolated from the sera of mice immunized with MSP119 inhibited
parasite growth in vitro in a dose-dependent manner, whereas the IgG from
pre-immune sera had no effect on parasite growth. At concentrations of 0.98
mg/ml, the antibodies isolated from mouse sera inhibited parasite growth by
75.7%.

Production of intracellular
cytokines

The fact
that C57BL/6 mice responded well after immunization with MSP119 suggested that
MSP119 contained epitopes recognized by T cells which provided help for
antibody production. To confirm that the MSP119 presented epitopes recognized
by mouse T lymphocytes, we evaluated the proliferative response and IFN-g
secretion of lymph node cells of C57BL/6 mice immunized with MSP119 emulsified
in CFA. We observed that upon in vitro stimulation with MSP119, these
cells proliferated and secreted IFN-g (Fig. 6).
The immune response was specific because lymph node cells from CFA-immunized
animals did not proliferate or secrete IFN-g. This result demonstrates that
MSP119 contains epitopes recognized by lymph node cells of C57BL/6 mice.

Discussion

Plasmodium
merozoites are major targets of blood-stage malaria vaccine development because
they are the only form that is exposed to the host immune system during this
stage. Intervention in merozoite invasion of erythrocytes would, in principle,
block the erythrocytic life cycle of the parasite and prevent the clinical
manifestations of infection. P. falciparum MSP119 is a very important
asexual blood-stage antigen, its right protein folding and the mild
post-translational modification are required for functionality [11,12]. The P.
pastoris system has the potential not only for high-level expression of
foreign genes but also for production of correctly folded, fully functional
products [13–15], which is induced by methanol and repressed by other carbon sources
such as glucose, glycerol, and ethanol [16]. Another important feature of this
system is its ability to achieve extremely high cell densities, enabling
efficient protein production and secretion [17].

Numerous
studies have demonstrated that humoral immunity plays a crucial role in the
protection against blood-stage malaria parasites [18,19]. Specific antibodies
to MSP119 can mediate the protective immunity, but it is necessary to induce
high levels of the antibodies [20–23]. As shown in this study, after
immunization of C57BL/6 mice with recombinant protein of MSP119 produced as a
secreted non-glycosylated polypeptide from P. pastoris, we observed that
protein MSP119 induced high levels of specific antibodies in mice after the
second immunization. The protective efficacy of humoral immunity in blood-stage
malaria was not only correlated to the level of IgG, but also associated with
isotypes of IgG. Many studies on serology conducted in malaria endemic regions
have demonstrated that cytophilic IgG1 and IgG3 were major isotypes that
associated with protective immunity in humans [24-27]. In this study, the
prevailing isotypes of IgG induced by MSPWe
also observed that upon in vitro stimulation with recombinant MSP119, mouse T
lymphocytes proliferated and secreted high levels of IFN-g. These results
suggest that the epitopes present in MSP119 were recognized by T lymphocytes
capable of providing antibody help. Diallo et al. [28] examined the levels and
distribution of IgG antibodies to MSPIn
summary, we have expressed recombinant MSP119 as a secreted non-glycosylated
polypeptide from P. pastoris. Antibodies against MSP119 can recognize the
native protein. The protein was highly immunogenic in all mice tested.

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