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* Cooperative Research Center for Vaccine Technology, Queensland Institute of Medical Research, Royal Brisbane Hospital, Queensland, Australia; and
Department of Microbiology, Faculty of Public Health, and
Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Merozoite surface protein 1
(MSP1)3 is a high
molecular mass (
185- to 205-kDa) glycoprotein expressed on
the surface of merozoites (2, 3, 4). It is a potential
vaccine candidate because it is directly exposed and interacts with the
host milieu during RBC invasion. The MSP1 precursor protein, which has
been shown to induce complete protection against Plasmodium
falciparum in monkeys (5), is processed by proteases
into a number of fragments. The primary processing at schizont rupture
cleaves the precursor protein into major fragments of 83
(MSP183), 30 (MSP130), 38
(MSP138), and 42 (MSP142)
kDa. The fragments are found as a noncovalently associated complex held
together on the free merozoite surface by the C-terminal membrane-bound
42-kDa fragment (2, 3, 4). At the time of merozoite invasion,
MSP142 is cleaved into two products. The soluble
33-kDa fragment (MSP133), corresponding to the
N-terminal region of MSP142, is shed from the
free merozoite surface (6). The membrane-bound 19-kDa
C-terminal fragment (MSP119), which contains two
epidermal growth factor-like domains (7), is the only
fragment carried with an invading merozoite into the new RBC
(8).
Early studies suggested that T cells specific for native MSP1 could protect mice independent of Ab (9). However, when MSP119, which can induce high levels of protection from homologous challenge in mice (10) and monkeys (11), was assessed for its ability to stimulate protective T cells, results were uniformly negative (12). Vaccination with T cell epitopes from MSP119 was unable to induce any level of protection and adoptively transferred T cells specific for either MSP119 or defined T cell epitopes did not render the recipients resistant to infection. It thus appeared that if T cell epitopes capable of protecting mice in the absence of Ab existed on MSP1, they must occur on fragments other than MSP119. Since MSP142 has been shown capable of protecting both monkeys (11, 13) and mice (14) from homologous challenge, we decided to examine MSP133 (which is contained within MSP142) for the presence of protective T cell epitopes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to 8-wk-old female BALB/c (H-2d), BALB/c nu/nu (nude), BALB/c SCID, and C57BL/6 (H-2b) mice were used. Animals were purchased from Animal Resources Center (Willetton, Western Australia, Australia) and were housed in the animal house under pathogen-free conditions. Nude and SCID mice were housed in filter top cages and were handled in laminar flow cabinets.
P. yoelii YM strain was used. The parasite was maintained by i.p. injection with 106 parasitized RBCs (pRBC) every 5 days.
Synthetic peptides and recombinant MSP119
Twenty-six peptides corresponding to
MSP133 (aa 1394–1657 (15)) were
produced at either the Queensland Institute of Medical Research
(Queensland, Australia) or Mimotopes (Clayton, Victoria, Australia).
Peptides were 20 aa in length, overlapping each other by 10 aa, and
were termed Cm 1–Cm 26 (Fig. 1
). The
purity of peptides was >85%, except peptide Cm 2, where crude peptide
was used.
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Lymphoproliferation assay and generation of T cell lines
Mice were immunized in hind footpads with Ags (30 µg of
peptide) emulsified in CFA (Sigma-Aldrich, St. Louis, MO). Nine to 10
days later, inguinal and popliteal lymph nodes were removed and
single-cell suspensions were prepared. Cells were washed with Eagle’s
MEM and were cultured in a volume of 200 µl in MEM supplemented with
50 µM 2-ME and 2% heat-inactivated normal mouse serum at 2 x
106 cells/ml in flat-bottom 96-well plates. Cells
were cultured with different concentrations of Ag (final concentration
of 30, 10, and 3 µg/ml) for 72 h and then were pulse labeled
with 0.25 µCi of [3H]thymidine. Incorporation
of radiolabel was estimated 18–24 h later by beta emission
spectroscopy. 
cpm were determined by subtraction of cpm in the
absence of Ag from cpm in the presence of Ag.
T cell lines specific to dominant epitopes or OVA were generated as described previously (17).
Immunization and challenge infection
Mice were immunized with PBS or Ags using a vaccination protocol described previously (10). Briefly, mice were immunized s.c. with PBS, 20 µg of peptides, or MSP119 in CFA. Some groups were vaccinated with the pool of dominant peptides at 20 µg of each peptides. The mice were then boosted four times with the same dose of Ag, s.c. in IFA (Sigma-Aldrich) at 21 days, i.p. in IFA at 42 and 56 days, and finally i.p. in PBS at 63 days. Ten days after the last immunization, the mice were challenged i.v. with 104 live P. yoelii YM pRBC. Parasitemia was monitored after infection by microscopic examination of smears from tail blood stained with Diff-Quick stain (Lab Aids, Narrabean, Australia).
Adoptive transfer study
A total of 107 viable resting T cells was purified by centrifugation over Ficoll-Paque, washed twice, and injected i.v. into nude or SCID mice. The mice were then challenged i.v. 4–24 h later with 104 P. yoelii YM pRBC.
Priming mice with T cell epitopes
Mice were primed s.c. with 20 µg of peptides emulsified in CFA. Two weeks later, the mice were immunized by infection and cured as described previously (12). Sera were collected to assess Ab responses.
Cell surface phenotype characterization
Single-cell suspensions of T cell lines were stained with PE- or
FITC- conjugated mAbs specific for mouse CD4, CD3, CD19, NK1.1 cells,
TCR
, and TCR
(Caltag Laboratories, Burlingame, CA). Cells
were incubated for 30 min at 4°C, washed twice with washing buffer
(0.1% BSA/0.1% sodium azide/PBS), and resuspended in 250 µl of 1%
paraformaldehyde. The percentage of positive cells was measured by a
FACS (BD Biosciences, San Jose, CA) and analyzed using CellQuest
software (BD Biosciences).
ELISA
Serum Ab levels against crude parasite Ags were analyzed by ELISA as described previously (12).
Western blot
Crude parasite Ag was separated on SDS-polyacrylamide gels (4% stacking and 12% separating gel; Bio-Rad, Hercules, CA) at 100 V for 1 h. Gels were electrophoretically blotted onto nitrocellulose paper, from which strips were cut and blocked overnight with 1 ml of PBS containing 5% skim milk. Strips were incubated with 1 ml of 1/100 mouse sera in 0.05% skim milk/PBS at room temperature for 2 h. After washing three times with 0.05% Tween 20/PBS, strips were incubated with 1 ml of 1/3000 goat anti-mouse Ig HRP conjugate (Silenus Labs, Melbourne, Australia) for 1 h at room temperature. Strips were washed three times, incubated with substrate (4-chloro-1-naphtol; Sigma-Aldrich) for 20 min, and washed three times with water.
Bioassay for IFN-, IL-2, and IL-4
Culture supernatants from T cell lines were collected 24, 48,
and 72 h after stimulation. IFN-, IL-2, and IL-4 activity were
determined as described previously (18). IFN- activity
was determined by measuring inhibition of WEHI-279 cell proliferation.
IL-2 and IL-4 activities were determined using the cytokine-dependent
cell lines CTLL-2 and CT.4S, respectively. The concentrations were
calculated from cytokine standards in the assays.
Statistics
Student’s t test for unpaired observations was used to determine differences between groups.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To define T cell epitopes on MSP133, BALB/c
mice or C57BL/6 were immunized in the footpads with pools of purified
peptides grouped as Cm1 and 3–6, Cm7–11, Cm12–16, Cm17–21, and
Cm22–26. Crude peptide Cm2 was used alone. Ten days after
immunization, draining lymph nodes were removed and tested for their
proliferative response in vitro to an individual peptide at three
different concentrations. Proliferative responses were assessed to be
significant at a stimulation index of 3 or above. BALB/c mice
recognized peptides Cm3, 4, 11, 15, and 21 (Fig. 2), whereas C57BL/6 mice recognized
peptide Cm11. Peptides Cm3 and Cm23 induced low-level proliferative
responses in C57BL/6 mice (stimulation index, <3) on two occasions
(Fig. 2, A and E) and peptide Cm23 induced
low-level responses in BALB/c mice (Fig. 2E), so these
peptides were selected along with the dominant epitopes for further
study.
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To determine whether the dominant T cell epitopes could induce
effector T cells capable of mediating protection against P.
yoelii infection, we used two approaches. First, normal BALB/c and
C57BL/6 mice were immunized with the peptides following the standard
vaccination protocol for MSP119 (10)
and were then challenged with P. yoelii YM. Control groups
were immunized with PBS or MSP119. BALB/c mice
that were immunized with PBS or individual peptides succumbed to
infection (Fig. 3A) with the
exception of one mouse that was immunized with Cm21 which developed
parasitemia, but gradually recovered. There was also one mouse
immunized with the pool of peptides (Cm3, 4, 11, 15, 21, and 23)
that developed parasitemia but recovered. All mice that were vaccinated
with MSP119 survived infection with one mouse
developing patent parasitemia. C57BL/6 mice that were vaccinated with
individual peptides developed parasitemia and died at the same rate as
the PBS control group (Fig. 3B). However, three of four mice
that received the pool of peptides (Cm3, 11, and 23) developed
parasitemia but were then able to control parasite growth. Positive
control mice that were immunized with MSP119 did
not develop detectable parasitemia. One representative mouse from each
group was sacrificed on the day of challenge to assess proliferation of
spleen cells induced by specific peptides used for immunization. In all
cases, spleen cells proliferated in response to specific peptides (data
not shown).
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The second approach used to establish the effector function of peptide-specific T cells was to adoptively transfer T cell lines specific for defined epitopes to naive nude mice. This approach was followed because we (19, 20) and others (21) have shown that T cells can adoptively transfer protection to malaria and because it is possible that the vaccination protocol (above) was unable to induce sufficient numbers of T cells. Furthermore, the phenotypes of vaccine-induced T cells is likely to be heterogeneous. The experiments were performed with T cells lines derived from BALB/c mice generated to peptides Cm3, Cm4, Cm11, Cm15, and Cm21.
T cell lines specific for dominant epitopes were generated by repeated
cycles of stimulation and rest in vitro. They were
CD3+, CD4+, and
TCR+ and produced IFN- and IL-2
following Ag-specific stimulation (Table I). All T cell lines proliferated
following stimulation with the immunogen (Fig. 4). Cm15- and Cm21-, but not Cm3-, Cm4-,
and Cm11-, specific T cell lines responded to whole parasite Ag. Nude
mice administered peptide-specific T cell lines were challenged with
P. yoelii YM and parasitemia was monitored. Nude mice that
received Cm3-, Cm 4-, and Cm11-specific T cells died within 11 days,
similarly to mice transfused with a T cell-line specific to an
irrelevant Ag, OVA (Fig. 5). However,
nude mice that were administered Cm15- or Cm21-specific T cell lines
demonstrated significantly suppressed parasite growth, indicating that
these epitopes were able to induce functional T cells. In these mice,
survival was prolonged from 8.5 to 21.5 days for mice that received
Cm15-specific T cells (p < 0.01) and from 8.5
to 29.8 days for mice that received Cm21-specific T cells
(p < 0.01). In addition, parasite density at
day 8 was reduced from 87.8 ± 2.7% to 19.2 ± 10.6% for
Cm15-specific T cell recipients (p < 0.01) and
from 87.8 ± 10.6% to 24.6 ± 11.2% for Cm21-specific T
cell recipients (p < 0.01). Sera taken from
recipient mice after challenge did not show detectable Ab to crude
parasite Ags as determined by ELISA and Western blot (Fig. 6).
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). In
contrast, the survival of Cm21-specific T cell-transfused SCID mice was
prolonged from 8.6 to 12.6 days (p < 0.02) and
parasite density at day 7 was reduced from 59.94 ± 7.51% to
10.56 ± 4.96% (p < 0.01).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MSP142, which gives rise to MSP133 and MSP119, has been shown to induce protective immunity in mice (14) and monkeys (11, 13). The degree of protection induced by MSP142 correlates with the level of specific Abs (11), and passive transfer of immune sera from immunized mice confers partial protection to the recipient animals (14). It has been shown that immune sera from animals immunized with MSP142 can inhibit parasite growth in vitro (13, 26). Preincubation of anti-MSP142 sera with MSP119 results in loss of the binding of anti-MSP142 sera to parasite MSP1 or MSP142. Furthermore, anti-MSP119-depleted sera failed to inhibit parasite growth in vitro, suggesting that inhibitory epitopes of MSP142 are localized on MSP119 (27). Sera from monkeys immunized with recombinant MSP142 recognize the 19-kDa fragment on Western blot (13). These data suggest that B cell epitopes of MSP142 are localized on MSP119. Studies of natural immune responses to MSP142 in humans have shown that B cell epitopes on MSP119 are recognized by immune sera from P. falciparum-exposed individuals, whereas the T cell proliferative responses are predominantly induced by the dimorphic regions of MSP133 (28). Similar observations by Egan et al. (29) have shown that T cell epitopes on MSP133 are commonly recognized by mononuclear cells from malaria-exposed individuals.
Although T cell responses against MSP133 have
been demonstrated (28, 29), their roles in immunity to
blood stage malaria remained unclear. In this report, T cell epitopes
on P. yoelii MSP133 were identified
and their roles in immunity to malaria were then studied. Using
overlapping peptides, we found that MSP133
contains multiple T cell epitopes. C57BL/6 mice recognized fewer
epitopes than BALB/c mice, suggesting that responses to
MSP133 are MHC class II restricted. Cm3 and Cm11,
which were recognized by both strains of mice, and Cm4, which was only
recognized by BALB/c mice, did not induce protection following
immunization with individual peptides. One mouse that received Cm21 was
able to clear parasitemia following challenge infection, suggesting
that Cm21 may be able to induce effector T cells capable of protection.
To confirm the effector role of T cells induced by
MSP133-dominant epitopes, T cell lines specific
to individual peptides were generated and were then transferred into
nude mice. Cm15 and Cm21 were of particular interest, as they were able
to confer partial protection against lethal P. yoelii YM to
recipient mice. It is probably relevant that these T cell lines were
the only ones to respond to whole parasite Ag in vitro. No
malaria-specific Abs were detected in these mice, suggesting that Cm15-
and Cm21-specific T cells are able to control parasite growth
independently of Abs. It is unlikely that T cell-transfused nude mice
developed their own Abs at levels below detection, since adoptive
transfer of T cells into SCID mice gave similar results. Since C57BL/6
mice vaccinated with the pool of peptides were better protected than
mice immunized with individual peptides, adoptive transfer of pooled T
cell lines may induce more effective protective immunity by ensuring
that mice have higher numbers of effector T cells at the time of
challenge. However, the mechanism of this enhanced immunity warrants
further study. Cm15- and Cm 21-specific T cell lines used in the
adoptive transfer study were of the Th1 type, producing IFN-
following Ag-specific stimulation in vitro. We noted that Cm11-specific
T cells also produced IFN- (as much as Cm21 cells and more than Cm15
cells) but did not induce protection. Cm15-and Cm21-specific T cells
proliferated in response to whole parasite Ags whereas Cm11 specific T
cells did not. IFN- and TNF are thought to be important in immunity
as are downstream molecules (30). However, the most
important correlate in our study was with the ability to recognize
whole parasites. Cm11 may be a "cryptic" epitope (31)
and although it may induce a IFN- response following peptide
stimulation, that is inconsequential if the cells cannot be activated
by parasites.
It is worth noting that passive transfer of an anti-MSP133 mAb recognizing MSP142 in merozoite extracts and the intact MSP1 precursor did not affect the course of parasitemia following infection with P. yoelii YM (32). This further suggests that the protection we have observed is Ab independent.
It is generally considered that immunity to P. yoelii is primarily Ab-mediated (25). Here, we demonstrate that T cell epitopes on MSP133 are able to induce effector T cells capable of controlling P. yoelii growth in an Ab-independent manner. Natural T cell responses to MSP133 are directed toward the conserved regions on P. falciparum MSP142 (28, 29). This along with data presented here suggests a strong case for further investigation of T cell epitopes of MSP133 as potential candidates for inclusion in a subunit vaccine. As cellular immune responses are crucial in mediating protection against many infectious diseases (33), incorporating epitopes that would induce effector T cells may enhance the efficacy of a vaccine against malaria.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael F. Good, Queensland Institute of Medical Research, P.O. Royal Brisbane Hospital, Queensland, 4029 Australia. E-mail address: michaelG{at}qimr.edu.au
3 Abbreviations used in this paper: MSP1, merozoite surface protein 1; pRBC, parasitized RBC.
Received for publication January 22, 2002. Accepted for publication May 7, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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