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Cooperative Research Centre for Vaccine Technology, Queensland Institute of Medical Research, P.O. Royal Brisbane Hospital, Brisbane; and
Walter and Eliza Hall Institute of Medical Research, P.O. Royal Melbourne Hospital, Melbourne, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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T cell depletion, can
cause a significant drop in antiparasite immunity in either immunized
normal or immunized B cell KO mice. In normal mice, this loss of
immunity is not accompanied by a decline in Ab levels. These
observations indicate a role for AMA1-specific Ab-independent T
cell-mediated immunity. However, the loss of immunity in normal
CD4+ T cell-depleted mice is temporary. Furthermore,
immunized B cell KO mice cannot survive infection, demonstrating the
absolute importance of B cells, and presumably Ab, in AMA1-induced
immunity. CD4+ T cells specific for a cryptic conserved
epitope on AMA1 can adoptively transfer protection to athymic
(nu/nu) mice, the level of which is enhanced by
cotransfer of rabbit anti-AMA1-specific antisera. Recipients of
rabbit antisera alone do not survive. Some protected recipients of T
cells plus antisera do not develop their own AMA 1-specific Ab
response, suggesting that AMA 1-specific CMI alone can protect mice.
These data are the first to demonstrate the specificity of any
protective CMI response in malaria and have important implications for
developing a malaria vaccine. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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90 different countries are at risk of developing the
disease, and up to 500 million cases of malaria occur each year
(1). This results in the deaths of 2–3 million people,
mainly children under 5 years of age, but also including a significant
number of pregnant women. Despite the availability of many intervention
strategies, estimates of morbidity and mortality continue to rise. This
reflects the reduced effectiveness of chemotherapy and vector control
programs due to the emergence and spread of insecticide-resistant
mosquito vectors and drug-resistant forms of Plasmodium
falciparum. An effective malaria vaccine is urgently needed
(2). The key to vaccine development is the identification of target Ags, development of processes to produce and purify these Ags, assessment of the immunological responses to them, and the ability of these responses to control parasite growth. Studies in rodent malaria models have facilitated our understanding of host-parasite interactions in vivo.
Apical membrane Ag 1 (AMA1)3 is a leading malaria vaccine candidate Ag which appears on the surface of merozoites after its release from the rhoptries (3). The AMA1 homologue for P. chabaudi has been cloned, and mice immunized with rPcAMA1 are afforded significant protection (4). Anti-AMA1 Ab titers are correlated with protection, and Abs, raised in rabbits, adoptively transferred protection (5); however, the mechanism of protection is not well understood. It is likely that immunity will be dependent not only on Abs present at the time of challenge but also on the continued synthesis of Abs post challenge, a state that is dependent on B cells but also on AMA1-specific Th cells that can be activated by the parasite. This has been demonstrated to be the situation in immunity induced by vaccination with the 19-kDa carboxyl-terminal segment of merozoite surface protein 1 (MSP119) (6, 7).
Although Abs are likely to be critical in immunity induced by AMA1, it
is also clear that effector T cells (
ß and ) can mediate or
contribute to immunity to malaria independently of B cells
(8, 9, 10, 11). In Plasmodium yoelii, early
experiments (12) showed that B cell-deficient mice were
unable to control infection. However, after drug cure, these mice were
capable of resisting a secondary challenge with homologous parasites,
indicating that resistance to reinfection was mediated at least in part
by an Ab-independent mechanism. Previous work from our laboratory has
demonstrated that effector T cells, in the absence of Ab, can control
parasite growth and, in some cases, eradicate rodent malaria parasites
(13, 14, 15). On the other hand, however, it has been
demonstrated that effector T cells specific for
MSP119 are unable to control parasitemia
(6, 7). It is thus curious that although
CD4+ T cells can control parasites in some
situations, this is not universally the case. An important role of T
cells specific for AMA1 in controlling P. chabaudi infection
may reside in helper function for Ab production (16, 17).
It is not known whether AMA1-specific effector T cells can control
infection.
This study explores the mechanisms of AMA1-mediated immunity, specifically examining the role of AMA1-specific T cells in controlling P. chabaudi adami infection, and demonstrates that Abs and effector T cells (operating through Ab-independent mechanisms) can control malaria in a murine model. We also present data showing that CD4+ T cells specific for a cryptic conserved T cell epitope on AMA1 are able to confer protection synergistically with AMA1-specific Ab, providing a strategy for the use of such epitopes in vaccine design.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female inbred BALB/c (H-2d), BALB/c-nu/nu and C57BL/6J (H-2b) mice were purchased from Animal Resource Centre (Perth, Australia). C57BL/6J µ chain knockout mice (18) were obtained from The Centenary Institute for Cancer Medicine and Cell Biology (Sydney, Australia) and bred in our animal facility. All mice ranged in age from 6 to 8 wk when experiments were initiated.
P. chabaudi adami strain DS parasites were used in our challenge experiments (5). The parasites were maintained by passage through donor mice.
Recombinant refolded AMA1 protein, AMA1 peptides, and rabbit anti-AMA1 sera
Refolded, Escherichia coli-expressed, recombinant
ectodomain of P. chabaudi adami (DS stain) AMA1 (denoted
rAMA1B) was produced as previously described (4). Peptides
(Table I
) (17) were
synthesized by the tea bag method (19) at the Queensland
Institute of Medical Research Peptide Unit with purity assessed by
reverse phase HPLC. Rabbit anti-AMA1 sera were generated as
previously described (5).
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Groups of three to five mice were immunized i.p. with 15 µg of
rAMA1B emulsified in Montanide ISA720. Four weeks later, a booster
immunization was given using the same amount of rAMA1B emulsified with
Montanide ISA720. Controls were immunized with PBS emulsified in
Montanide ISA720. Ten days later the mice were challenged i.v. with
1 x 105 P. chabaudi
adami parasitized erythrocytes. In some studies, mice
were depleted of CD4+ T cells or T cells
before challenge (see below). Parasite densities were monitored by
microscopic examination of tail blood films during the course of
infection.
In vivo CD4+ and T cell depletion
For depleting CD4+ or T cells, mice
were given three daily i.p. injections of either 1 mg rat anti-CD4
(GK1.5) or 0.5 mg hamster anti- T cells (GL3) (gift of Dr.
Jean Langhorne), purified from ascites by ammonium sulfate, before
challenge, and then once per week during the course of infection.
Control mice received normal rat IgG. The success of depletion was
confirmed by flow cytometric analysis of peripheral blood (PB) samples
of each mouse.
Flow cytometry analysis
For analysis of depletion of CD4+ T cells
or T cells, 1 x 106 mononuclear
cells from PB were incubated with a 1:50 dilution of FITC-conjugated
rat anti-mouse CD4 (Caltag, South San Francisco, CA) or a 1:50
dilution of FITC-conjugated hamster anti-mouse T cells
(Caltag) for 30 min at 4°C. After a washing with buffer (0.1%
BSA-PBS), cells were fixed in 1% paraformaldehyde in PBS. For analysis
of T cell lines, rested T cells were incubated at 4°C for 30 min with
undiluted hybridoma supernatants. After two washes, cells were
incubated for an additional 30 min at 4°C with FITC-labeled goat
anti-rat IgG or goat anti-hamster IgG (Caltag), washed, and
fixed in 1% paraformaldehyde in PBS. Fluorescence was analyzed using a
FACScalibur flow cytometer (Becton Dickinson, Mountain View, CA) with
the use of the Cellquest program. For each sample, 10,000 events were
counted, and the percentage of positive cells was determined after
correction for nonspecific fluorescence.
The following rat IgG mAbs were used for staining: anti-CD3 (KT3),
provided by Dr. T. Mandel (The Walter and Eliza Hall Institute of
Medical Research, Melbourne, Australia); anti-CD8 (53-6.72,
American Type Culture Collection (ATCC), Manassas, VA); anti-B220
(purchased from ATCC); and anti-CD4 (GK1.5), provided by Professor
A. Kelso (Queensland Institute of Medical Research). Hamster IgG mAbs
included anti-TCRß (H57-597) provided by Dr. T. Mandel, and
anti-TCR (GL3-1A) provided by Dr. K. Shortman (The Walter and
Eliza Hall Institute of Medical Research, Melbourne, Australia).
Ab assay
AMA1-specific Abs in sera were measured using ELISA. Briefly, polyvinyl chloride plates (ICN Biochemicals Australia, Sydney, Australia) were coated overnight at 4°C with 0.2 µg/ml rAMA1B. After coating and between each incubation step, plates were washed three times with PBS-Tween 20. Plates were blocked for 1 h at 37°C with 1% BSA in PBS/Tween 20 before samples of serum diluted in PBS/Tween 20 were added to individual wells. After a 1-h incubation at 37°C, 100 µl of a 1:3000 dilution of peroxidase-conjugated sheep anti-mouse IgG or anti-IgG isotype Ab (The Binding Site, Birmingham, U.K.) were added for 1 h at 37°C, followed by the addition of 100 µl/well of 2,2'-azino-di-(ethyl-benzthiozoline sulfonate) (Sigma, St. Louis, MO). After 30 min, the absorbance of each well was read at 405 nm. Serially diluted sera were used to determine Ab titers. For isotype determination, 1:500 diluted sera were used.
Generation of CD4 peptide-specific T cell lines
T cell lines were generated from the lymph node cells of mice immunized with a pool of P4 and P5 peptides. Briefly, the mice were immunized in the hind footpads with 10 µg pooled P4/P5 peptides (5 µg each) emulsified in CFA (H37Ra, Difco, Detroit, MI). From 7 to 9 days postimmunization, draining inguinal and popliteal lymph node cells were removed and suspended at 2 x 106 cells/ml in culture medium consisting of Eagle’s MEM (EMEM, Trace Biosciences, Victoria, Australia) supplemented with 10% FCS, 100 U/ml benzyl penicillin (Commonwealth Serum Laboratories, Melbourne, Australia), and 5 x 10-5 M 2-ME (Sigma). Cells were dispensed in 24-well cluster plates (Corning Glass Works, Corning, NY) containing 5–10 µg/ml peptides. After 4 days, viable cells were isolated on Ficoll-Paque (Pharmacia LKB Biotechnology, Uppsala, Sweden), washed, and dispensed in 24-well plates containing irradiated (2500 rad) syngeneic spleen cells (APCs) at 2 x 106/ml in the absence of Ag for 7–10 days (rest phase). For expansion, the rested cells were restimulated with peptide in the presence of APCs (2 x 106 cells/well). The phenotype of T cells was determined by FACS analysis as described above. The specificity of T cells was assessed by T cell proliferation assays.
T cell proliferation assay
Lymph node cells. Seven to nine days after immunization, the popliteal and inguinal lymph nodes were removed and pooled, a single-cell suspension was prepared, and the cell concentration was adjusted to 2 x 106 cells/ml in proliferation medium consisting of EMEM, 2% syngeneic normal mouse serum, 100 U/ml benzyl penicillin, and 5 x 10-5 M 2-ME. This suspension (200 µl) was added to 96-well flat-bottom plates (Nunclon, Nunc, Copenhagen, Denmark) containing appropriate Ag.
T cell lines. After 7–10 days of rest, viable T cells were isolated by Ficoll-Paque gradient centrifugation, washed, and resuspended at 1 x 106 cells/ml in proliferation medium. This suspension (100 µl) was added to 96-well flat-bottom plates containing 1 x 106 APCs and appropriate Ag.
All assays were for 4 days. [3H]Thymidine (DuPont, Boston, MA; 0.5 µCi in 25 µl EMEM) was added to each well for the final 12–18 h of culture. Cells were harvested onto filter mats (Wallac Oy, Turku, Finland), and incorporated radioactivity was determined in a liquid scintillation counter. Results are expressed as the mean cpm ± SD.
Adoptive transfer and challenge experiments
T cells were harvested after 7–10 days rest in bulk culture and enriched over a Ficoll-Paque gradient (400 x g for 20 min). Viable T cells (1 x 105) were resuspended in 200 µl of PBS and injected i.v. via the lateral tail vein into BALB/c-nu/nu mice. Some groups of mice also received three daily i.p. injections of 0.125 ml rabbit anti-AMA1 sera, beginning on day 0 of infection. Mice were challenged i.v. with 1 x 105 P. chabaudi adami-infected RBC (pRBC) 24 h after T cell transfer. Parasite densities were monitored as described above.
Statistical analysis
Comparisons among experimental groups by Student’s t test were done using a statistical analysis program of Sigma Plot for window version 4.0 (SPSS). Significance was set at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To demonstrate protection after rAMA1B immunization, BALB/c and
C57BL/6J mice were immunized and boosted with recombinant P.
chabaudi rAMA1B protein combined with the adjuvant, Montanide
ISA720. Ten days after immunization, mice were challenged with pRBC,
and the parasitemias were monitored every second day. As shown in Fig. 1, both BALB/c and C57BL/6J immunized
mice demonstrated significantly lower peak parasitemias compared with
PBS-immunized mice (5.1 ± 4.5 vs 25.8% ± 11.3 in BALB/c mice,
p < 0.05; 13.2 ± 14.3% vs 57.7% ± 6.8 in C57
BL/6J mice, p < 0.01). These results confirmed our
previous studies, which showed that rAMA1B immunization can induce
significant protection to P. chabaudi adami infection
(5). rAMA1B-immunized C57BL/6J mice had significantly
higher peak parasitemia than rAMA1B-immunized BALB/c mice (Fig. 1, p < 0.05).
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To determine whether CD4+ T cells are critical to immunity after rAMA1B immunization, CD4+ T cells were depleted from BALB/c mice postimmunization by treatment with anti-CD4 Abs. Control animals were treated with control Abs (see Materials and Methods). The success of depletion was checked by FACS analysis on PBL of each mouse which showed that > 99% of CD4+ T cells were depleted at the time of challenge (data not shown).
rAMA1B-specific Ab production. High
titers (2,048,000 ± 0) of AMA1-specific Abs were detected in both
strains of immunized mice before challenge. Titers increased by day 2
postchallenge (5,461,333 ± 1,366,952) in BALB/c mice and
2,069,733 ± 672,028 in C57BL/6J mice, respectively) and reached
the highest level (8,176,000 ± 5,144,217) in BALB/c mice and
6,848,000 ± 2,833,641 in C57BL/6J mice, respectively)) by day
four after challenge to then maintain similar levels over the period of
infection (Fig. 2). In PBS-immunized
mice, initial moderate titers (4333 ± 2029) of AMA1-specific Abs
were detected by day 14 postinfection, and these remained at similar
levels over the course of infection (Fig. 2A). There is
no significant difference in titer of IgG isotypes between
immunized/depleted and nondepleted mice (Fig. 2B).
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). Infection of nonimmunized
mice did give rise to moderate titers which were slightly reduced in
CD4+ T cell-depleted mice from day 14
postinfection.
Protection. After depletion of
CD4+ T cells, rAMA1B-immunized mice failed to
control parasitemia during a 6-day period (days 6–12), suffering
peak parasitemias of 38.43 ± 7.45% at day 10 postinfection
compared with 1.47 ± 1.47% in AMA1-immunized mice treated
with normal rat IgG (p < 0.01; Fig. 3). Mice were then able to control
parasitemia as well as undepleted controls. Mice immunized with PBS and
depleted CD4+ T cells suffered the highest peak
parasitemia levels (data not shown).
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T cells in B cell knockout mice after rAMA1B immunization
To determine whether the CD4+ T cells
specific for AMA1 were contributing to Ab-independent CMI, B cell
knockout (B-KO) mice and congenic controls (C57BL/6J) were immunized
with rAMA1B. We confirmed our previous results showing that
immunologically intact mice (C57BL/6J mice) were able to control
parasitemia after rAMA1B-immunization (data not shown). With the same
immunization protocol, rAMA1B-immunized B-KO mice had significantly
lower parasitemias over the course of infection than did PBS-immunized
B-KO mice (p < 0.05, Fig. 4). In contrast, when rAMA1B-immunized
B-KO mice were depleted of CD4+ T cells, the mice
suffered a significantly higher parasitemia compared with
rAMA1B-immunized control B-KO mice or rAMA1B-immunized B-KO mice
treated with normal rat IgG (p < 0.05; Fig. 4). Parasite densities in CD4+ T cell-depleted B
cell KO mice were similar to densities in PBS-immunized (nondepleted)
mice. However, all B-KO mice died with high parasitemia by day 12.
These results suggest that AMA1-specific CD4+ T
cells do contribute to the control of parasitemia in P. chabaudi
adami infection via an Ab-independent mechanism; however, Ab is
critical to protection.
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T cell depletion did not alter the course of
parasitemia in rAMA1B-immunized B cell KO mice (Fig. 4). AMA1-specific cryptic CD4+ T cells confer a protective role in P. chabaudi adami infection and synergize with AMA1-specific Abs
CD4+ T cell epitopes on P. chabaudi AMA1 have been defined previously (16). One epitope of particular interest, defined by two overlapping 20-mer peptides, P4 and P5, was shown to be cryptic and was in a highly conserved region of the protein. This cryptic epitope, identified by immunizing mice with synthetic peptides, was not revealed after immunization with rAMA1B; however, T cells induced by P4/P5 imunization were capable of responding in vitro to rAMA1B stimulation. Adoptively transferred T cells specific for this epitope were able to prevent 50% of BALB/c athymic (nu/nu) mice from succumbing to P. chabaudi adami.
A T cell line was generated to P4/P5 (see Materials and
Methods). After four cycles of stimulation and rest, the cell line
was characterized by FACS analysis, which showed it to be composed of
99% CD4+ T cells with 99% TCRß usage. The
specificity of the line was confirmed by T cell proliferation analysis
(data not shown).
To determine the potential of T cells specific for this cryptic
epitope to control parasitemia and their ability to synergize with
AMA1-specific Abs, 1 x 105 viable resting T
cells, rabbit anti-AMA1 antisera (3 x 125-µl doses), or
both, were transferred to BALB/c-nu/nu mice 24 h before
challenge (for T cells) or at days 0, 1, and 2 relative to the day of
challenge (for Ab). The onset of patent parasitemias was delayed in
mice receiving rabbit anti-AMA1 antiserum compared with mice
receiving normal rabbit serum, but both groups of mice died, usually at
high parasitemia (Fig. 5). Of the four
mice that received the T cells alone, two survived, with one
demonstrating a significant increase in AMA1-specific Abs 2 wk
postchallenge. The two mice that died experienced high parasitemia
(>60%), whereas the survivors had peak parasitemias of 1 and 18%. Of
the four mice that received both T cells plus antisera, three survived,
with one succumbing at a low parasitemia (1%). Three experienced peak
parasitemias of between 19 and 23%. Two of the three survivors
demonstrated a boost in AMA1-specific Abs at the time of the
recrudescence of parasitemia (day 40). We have compared peak
parasitemia and overall survival in mice that received T cells plus
antiserum vs antiserum alone and demonstrated a significant effect of T
cells on both parasitemia and survival (p <
0.01, and p < 0.01 respectively). These data suggest
that CD4+ T cells and specific Abs can act
synergistically. We also observed that three of the mice that did
survive and that received T cells (mice 1, 4, and 7) did so without the
development of a significant murine AMA1-specific Ab response.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells were shown to play no role in
immunity.
The evidence for an antiparasite effect mediated by
CD4+ T cells acting independently of B cells
comes from observing: 1) a significant diminution of antiparasite
control during a 6-day period after CD4+ T cell
depletion in immunized normal mice in which there was no diminution of
anti-AMA1-specific Ab levels; and 2) significantly heightened
parasite densities in immunized B cell KO mice that had been depleted
of CD4+ T cells. Because these mice had no B
cells, T cells could not function as helpers for Ab production, and
effects arising as a result of CD4+ T cell
depletion can be attributed to an effect on CMI. These data were
supported by observations that adoptively transferred cultured
CD4+ T cells could control parasite densities in
nude mice, some of which did not experience a rise in AMA1-specific
Abs. We could not exclude, however, the possibility that Abs of other
specificities (not measured) were contributing to the ultimate
protection in these mice, although that would seem unlikely given that
the AMA1-specific T cells would need to operate as helpers for B cells
specific for a different chabaudi Ag.
CD4+ T cells specific for another leading malaria
vaccine candidate, MSP119, cannot adoptively
transfer resistance to P. yoelii, and vaccination with
defined T cell epitopes from the yoelii homologue could not
delay patency, the time of death, or the peak parasitemia after
challenge (6). It is thus curious that
CD4+ T cells acting independently of B cell
helpers, although shown to be capable of protecting mice from malaria
in the absence of Ab in various systems when generated in response to
the whole parasite (e.g., 15), can play only an ancillary role in
immunity induced by AMA1 and have no effect when specific for
MSP119. Nevertheless, this is the first
demonstration of the specificity of a malaria CMI response, albeit one
that also requires the presence of an Ab response. We have not
determined the mechanism of action of AMA1-specific
CD4+ effectors, although TNF-- reactive
nitrogen intermediates and/or oxygen radicals would be expected to play
important roles (20). Preliminary data show that IFN-
was not produced in response to vaccination and challenge.
Although depletion of CD4+ T cells
postimmunization led to a temporary loss of immunity (as judged by
peripheral blood parasite densities (Fig. 3)), depletion did not result
in a diminution of AMA1-specific Abs after infection (Fig. 2). In fact,
the levels of Abs were nearly identical in both depleted and
nondepleted vaccinated mice. These data are similar to those presented
by Langhorne et al. (21) which showed that depletion of
CD4+ T cells 20 days after primary infection with
P. chabaudi adami did not prevent the IgG Ab titer rising
further into the infection, possibly because a few CD4 T cells remained
after depletion.
rAMA1B-immunized C57BL/6J mice had significantly higher peak
parasitemia than rAMA1B-immunized BALB/c mice (Fig. 1). This may result
from genetic control of anti-rAMA1B responsiveness. It could be
interesting to study Ab responsiveness in congenic strains of mice
differing only at H-2. It has also been demonstrated that BALB/c mice
have a bias toward Th2 responses (22, 23), which may also
explain our observed differences.
P. chabaudi is considered an example of a Plasmodium species in which CMI is critical for protection (9, 24, 25). The data here, however, point to the need for Abs to control infection and prevent death. Cell-mediated responses are capable of contributing to the overall control exerted by Abs but are not capable of preventing death when induced by vaccination and when acting alone. Overall, the data are consistent with previous data showing that denatured AMA1B can reduce mortality in mice challenged with P. chabaudi adami, although protection is less than that induced by vaccination with correctly folded AMA1B (5). Both folded and denatured AMA1B would be expected to activate T cells; however, only correctly folded material would induce appropriate Abs.
We have previously shown that T cells specific for cryptic
epitopes on chabaudi (P4/P5) can adoptively transfer partial
resistance to infection (16). In those experiments,
approximately one-half of the animals succumbed to infection. The
mechanism of enhanced protection was not defined, but there was some
evidence of a more rapid Ab response after infection in mice that
received P4/P5-specific T cells. We have extended those observations
here by coadministering to some mice rabbit anti-AMA1B-specific
antisera with the specific T cells. The rabbit antiserum kept parasite
densities lower, thus enabling the animals to survive longer and
allowing us to better monitor murine AMA1B-specific Abs arising as a
result of infection. Again we were able to show that one-half of the
nude mice that received the T cells alone survived; however, better
control of parasitemias was recorded for the group that received both T
cells and rabbit antisera. All mice that received rabbit anti-AMA1
antiserum or normal rabbit serum alone died. We observed that of the
four mice that received both T cells and rabbit anti-AMA1
antiserum, only one animal (mouse 2) developed a significant murine
anti-AMA1 response. The transfused Abs may have suppressed the
development of such a response, as was shown to occur in a different
malaria system (25, 26). The combination in nude mice of
this passively administered CMI response with a passively administered
anti-AMA1 Ab response gives rise to a degree of protection similar
to that which follows AMA1 vaccination of euthymic mice (Fig. 1).
This study does have implications for developing malaria vaccines. For two separate merozoite surface Ags (AMA1 and MSP119), Ab has now been shown to be critical and the Ab titer seems of paramount importance (5, 7). Adjuvants must be chosen that augment an Ab response. However, the other implication is that if CMI can contribute to parasite control in human malaria, it may be possible to design a vaccine that includes T cell epitopes from various Ags and is chosen on the basis of sequence conservation and HLA restriction by common alleles. Many polymorphic malaria Ags (including AMA1) have highly conserved segments, perhaps in part because certain segments may not be immunodominant with respect to Ab recognition.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. M. F. Good, The Queensland Institute of Medical Research, P.O. Royal Brisbane Hospital, Queensland 4029, Australia.
3 Abbreviations used in this paper: AMA1, apical membrane Ag 1; MSP1, merozoite surface protein 1; PB, peripheral blood; EMEM, Eagle’s MEM; pRBC, P. chabaudi adami-infected RBC; B-KO, B cell knockout; CMI, cell-mediated immunity.
Received for publication October 13, 1999. Accepted for publication April 19, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells contribute to control of chronic parasitemia in Plasmodium chabaudi infections in mice. J. Immunol. 162:2837.This article has been cited by other articles:
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  J. M. Burns Jr., P. R. Flaherty, P. Nanavati, and W. P. Weidanz Protection against Plasmodium chabaudi Malaria Induced by Immunization with Apical Membrane Antigen 1 and Merozoite Surface Protein 1 in the Absence of Gamma Interferon or Interleukin-4 Infect. Immun., October 1, 2004; 72(10): 5605 - 5612. [Abstract] [Full Text] [PDF]
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A. Rainczuk, T. Scorza, P. M. Smooker, and T. W. Spithill Induction of Specific T-Cell Responses, Opsonizing Antibodies, and Protection against Plasmodium chabaudi adami Infection in Mice Vaccinated with Genomic Expression Libraries Expressed in Targeted and Secretory DNA Vectors Infect. Immun., August 1, 2003; 71(8): 4506 - 4515. [Abstract] [Full Text] [PDF]
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M. S. Mueller, A. Renard, F. Boato, D. Vogel, M. Naegeli, R. Zurbriggen, J. A. Robinson, and G. Pluschke Induction of Parasite Growth-Inhibitory Antibodies by a Virosomal Formulation of a Peptidomimetic of Loop I from Domain III of Plasmodium falciparum Apical Membrane Antigen 1 Infect. Immun., August 1, 2003; 71(8): 4749 - 4758. [Abstract] [Full Text] [PDF]
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 M. O. Makobongo, G. Riding, H. Xu, C. Hirunpetcharat, D. Keough, J. de Jersey, P. Willadsen, and M. F. Good The purine salvage enzyme hypoxanthine guanine xanthine phosphoribosyl transferase is a major target antigen for cell-mediated immunity to malaria PNAS, March 4, 2003; 100(5): 2628 - 2633. [Abstract] [Full Text] [PDF]
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M. C. Kennedy, J. Wang, Y. Zhang, A. P. Miles, F. Chitsaz, A. Saul, C. A. Long, L. H. Miller, and A. W. Stowers In Vitro Studies with Recombinant Plasmodium falciparum Apical Membrane Antigen 1 (AMA1): Production and Activity of an AMA1 Vaccine and Generation of a Multiallelic Response Infect. Immun., December 1, 2002; 70(12): 6948 - 6960. [Abstract] [Full Text] [PDF]
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A. W. Stowers, M. C. Kennedy, B. P. Keegan, A. Saul, C. A. Long, and L. H. Miller Vaccination of Monkeys with Recombinant Plasmodium falciparum Apical Membrane Antigen 1 Confers Protection against Blood-Stage Malaria Infect. Immun., December 1, 2002; 70(12): 6961 - 6967. [Abstract] [Full Text] [PDF]
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 H. Xu, J. Wipasa, H. Yan, M. Zeng, M. O. Makobongo, F. D. Finkelman, A. Kelso, and M. F. Good The Mechanism and Significance of Deletion of Parasite-specific CD4+ T Cells in Malaria Infection J. Exp. Med., April 1, 2002; 195(7): 881 - 892. [Abstract] [Full Text] [PDF]
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) were measured in the sera as
described in Materials and Methods. + indicates that the
mouse died from infection.