Malaria is one of the most dreadful diseases caused by a protozoan parasite,
Plasmodium that has proved to be a hinderance to the cultural and socio-economic
progress of man in tropical and subtropical regions of the world. Resurgence
of malaria resulted due to increased resistance to conventional antimalarial
agents, both chemotherapeutic and chemoprophylactic, absence of an effective
malaria vaccine and weakening of malaria control programme. Due to lack of availability
of funds, future of malaria control programme has become uncertain (WHO,
The antigens of parasite play an important role in malaria control. An exposure
of the host with parasite antigens for longer duration activates both the humoral
and cell-mediated immune responses. Although many antigens of parasite have
been identified but a large number of them still remain unexplored which may
impart long lasting immunity. In this study, the cytosolic constituents of P.
berghei obtained after centrifugation at 20,000 rpm from asexual erythrocytic
stages of parasite exhibited humoral response in mice and effectively inhibited
in vitro propagation of parasite.
MATERIALS AND METHODS
Maintenance of parasite: The asexual erythrocytic stages of Plasmodium
berghei (NK-65), a rodent malaria parasite, were maintained in white Swiss
mice, Mus musculus, BALB/c strain with permission of Institutional Animal
Ethics Committee. The parasite was maintained in mice of either sex of about
20-25 gm weight, by passing the blood from infected mice to naïve intraperitoneally
(i.p.) so that each mouse gets 1x105 P. berghei infected
erythrocytes in citrate saline (Banyal et al., 1991).
Isolation of cell free Plasmodium berghei: Blood was collected
from P. berghei infected mice for the isolation of cell-free parasite.
When the percent infection was more than 50%, the mice were anaesthetized with
diethyl ether. Blood was collected by jugular vein incision in citrate saline
in the ratio of 2:1. Cell-free P. berghei was isolated according to the
method of Banyal and Fitch (1982).
Differential centrifugation: Differential centrifugation of the homogenized
parasite was carried by centrifugation at 20,000 rpm (Sigma 3K 30) for 35 min
at 4°C. The pellet was discarded
and the supernatant was used for immunization.The supernatant was designated
Protein estimation: Protein was estimated spectrophotometrically using
bovine albumen as standard (Banyal and Kumar, 1994).
Immunization of mice and collection of sera: A group of 10 mice was
immunized with fraction S. Each mouse was given 100 μg of protein along
with 30 μg saponin as adjuvant in 0.01 M PBS, pH 7.2 intraperitoneally
on day 0,14 and 28. Placebo controls received only saponin (30 μg mouse-1
dose-1) in PBS. A week after the last immunization dose all the mice
were sacrificed, sera collected separately and stored at -20°C
till further use.
Enzyme linked immunosorbent assay (ELISA): Enzyme linked immunosorbent
assay (ELISA) was performed according to Banyal and Inselburg
(1985) in 96 well polystyrene microtitre plates.
Indirect fluorescent antibody (IFA) test: Indirect Fluorescent Antibody
(IFA) test was carried according to Collins and Skinner
(1972) as given by Upma and Banyal (1998).
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE):
The antigens were analysed to determine their molecular weights by SDS-PAGE
according to Kumar and Banyal (1997).
In vitro merozoite invasion inhibition assay: The short term
in vitro culture of P. berghei was carried out as given by Upma
and Banyal (1998).
Culture medium: RPMI-1640 (Gibco) was used as culture medium. It was
supplemented with 0.06% (w/v) N-2-hydroxyethyl piperazine-N-2-ethane
sulphonic acid (HEPES): 5% (w/v) sodium bicarbonate; antibiotics gentamycin
(50 μg mL-1), penicillin (100 μL mL-1) and streptomycin
(100 μg mL-1). The pH of incomplete medium was adjusted to 7.4,
it was filtered through 0.22 μ millipore syringe filter under sterile conditions.
Ten percent (v/v) inactivated Foetal Calf Serum (FCS) was added to incomplete
medium and the complete medium was prepared.
Invasion inhibition assay: The invasion inhibition assay was carried
out in 12 well culture trays (TPP, Switzerland). To each well 1 mL of complete
medium having 3% haematocrit and 0.3-0.8% parasitaemia along with 50 μL
of normal/immune mouse serum was added. Each sample was run in duplicate. The
trays were then shaken gently to mix contents. The sample was taken, centrifuged
and from resulting pellet portion 0 h smears were prepared. The culture trays
were placed in a candle jar at 37°C. Culture trays were kept for 21 h in
the incubator, then removed from the incubator and smears from each well were
prepared after centrifugation of different samples. Smears were fixed in methanol
and stained with Giemsa stain. A differential count of the parasite (rings,
trophozoites and schizonts) in smears was done. The merozoite invasion inhibition
percent was calculated as:
Immunization of mice with fraction S resulted in appearance of antimalarial
antibodies. Indeed sera of ten mice the ELISA titre was 1:1024 in three mice,
1:2048 in three mice,1:4096 in two mice, 1:8192 and 1:16384 in one mouse each
(Fig. 1). Immunoflorescence reactions were observed in immune
sera as observed under UV light (Fig. 2).The short term in
vitro invasion inhibition assay revealed a significant inhibition of propagation
of P. berghei in vitro in the presence of immune sera. After 21 h of
incubation a significant number of rings appeared in the normal sera culture
(Table 1 ). In the sera of five out of ten mice immunized
with fraction S of P. berghei more than 50% inhibition of propagation
of parasite was observed. The number of newly formed rings in the experimental
culture compared to the normal control culture was significantly less. Table
1 also revealed that there was maturation of parasite in the in vitro
Sds page: The S fraction was subjected to SDS PAGE analysis. There were
18 protein bands ranging from 23 to 205 kDa (Fig. 3).
In vitro invasion inhibition assay: The parasite inhibitory property
of antimalarial antibodies present in sera from immunized mice were demonstrated
by short term in vitro invasion inhibition assay. P. berghei infected
red cells mainly consisting of trophozoite and schizonts were incubated along
with normal red cells in RPMI-1640 supplemented with foetal calf serum. After
21 h of incubation there was a increase in rings as compared to 0 h in the control
normal serum culture.
|| Merozoite invasion inhibition in vitro in the presence
of immune sera
||Histogram showing level of antibodies in immune sera of mice
analysed by ELISA
||IFA reaction seen under UV light and phase contrast (χ1000),
(a) Immune serum of immunized mouse (UV) and (b) Immune serum of immunized
mouse (Phase contrast)
||SDS page lane, (a) Molecular weight marker, (b) Total parasite
homogenate and (c) Fraction S
Effect of immune sera: The in vitro invasion inhibition assay
was performed using the sera from mice immunized with S fraction of parasite.
The smears prepared from culture supplemented with immunized sera after 21 h
of incubation showed that the number of trophozoites and schizonts decreased
as compared to 0 h and the number of rings in the experimental were less compared
to the control. The percent invasion inhibition observed with serum of 10 mice
as 32.25, 38.70, 54.83, 58.20, 52.00, 35.48, 22.5, 19.35, 51.61 and 51.61% (Table
Plasmodium berghei (NK-65) generally proved lethal to white Swiss mice
(BALB/c) has been confirmed in the present investigation. The immunization of
mice with P. berghei results in induction of both the humoral and cell-mediated
immune responses BALB/c strain of white Swiss mice. The present work investigated
the role of S (supernatant of 20,000 rpm) fraction of P. berghei in providing
immunity. This fraction exhibited a strong humoral immune response. Serodiagnostic
tests like IFA and ELISA were conducted. The increase in antibody level may
be due to activation of memory cells which released antiparasite antibodies
in blood. This shows that antigens present in S subcellular fraction of parasite
triggered the humoral response. The immune sera obtained from immunized mice
were further used for in vitro invasion-inhibition assay. The results
of in vitro invasion-inhibition assay showed inhibition of parasite invaded
to different levels.
Since this fraction contains 18 bands of molecular weight ranging from 23.15-205.00
kDa, it appears that some of these proteins have acted as antigen. Three antigens
of molecular weight 30, 40, 105 kDa have been reported from asexual blood stages
of P. falciparum which are inhibitory in vitro (Banyal
and Inselburg, 1985). In another study, antigens of molecular weights 80
and 95 kDa have been shown to induce strong humoral response as evident by IFA
and in vitro inhibition of merozoite invasion. Eighty three kDa protein
could be the apical membrane antigen (AMA-1), another leading vaccine candidate
which is a target for antibody dependent immune response and reported in all
Plasmodium species. AMA-1 in combination with MSP-1 have also been evaluated
for its efficacy against blood-stage malaria in animal models (Burns
et al., 2003). Unavailability of effective antimalarial vaccine has
become one of the major cause of immense suffering and despite extensive efforts
malaria has not been completely eradicated. Most malaria resurgences have been
linked to weakening of control programme (Cohen et al.,
From the in vitro culture studies it was found that S fraction exhibits
a strong humoral response and sera from mice immunized with this fraction showed
in vitro inhibition against malaria invasion, this fraction can be used
as candidate for effective malaria vaccine. The antigens of parasite need to
be explored further so as to know their protective role against malaria or their
cross reactivity with other antigens of simian or human malaria parasites.
Ms Monica Nainta is grateful to DST (New Delhi) for Inspire fellowship.