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Infection and Immunity, October 2005, p. 6363-6371, Vol. 73, No. 10
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.10.6363-6371.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Alteration in Host Cell Tropism Limits the Efficacy of Immunization with a Surface Protein of Malaria Merozoites{dagger}

Qifang Shi, Amy Cernetich, Thomas M. Daly, Gina Galvan, Akhil B. Vaidya, Lawrence W. Bergman, and James M. Burns Jr*

Center for Molecular Parasitology, Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19129

Received 23 March 2005/ Returned for modification 4 May 2005/ Accepted 26 May 2005


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ABSTRACT
 
Immunization with Plasmodium yoelii merozoite surface protein-8 (PyMSP-8) has been shown to protect mice against lethal P. yoelii 17XL malaria. Here we demonstrate that PyMSP-8-specific antibodies preferentially suppress P. yoelii 17XL growth in mature erythrocytes compared to growth in reticulocytes and do not suppress the growth of nonlethal P. yoelii 17X, a parasite that primarily replicates in reticulocytes. The protection against normocyte-associated P. yoelii malaria parasites is mediated by antibodies that recognize conformational epitopes of PyMSP-8 that are nonpolymorphic. We examined changes in gene expression in reticulocyte-restricted P. yoelii 17XL parasites that escaped neutralization by PyMSP-8-specific antibodies using P. yoelii DNA microarrays. Of interest, Pymsp-8 gene expression decreased, while the expression of msp-1, msp-7, and several rhoptry protein genes increased. Breakthrough parasites also exhibited increases in the expression of a subset of yir and Pyst-a genes that are predicted to encode polymorphic antigens expressed on the surface of infected erythrocytes. These data suggest that changes in the expression of parasite proteins expressed on the merozoite surface, as well as the surface of infected erythrocytes, may alter host cell tropism and contribute to the ability of malaria parasites to evade merozoite-specific, neutralizing antibodies.


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INTRODUCTION
 
Malaria is caused by protozoan parasites belonging to the genus Plasmodium. Clinical disease occurs when parasites invade and replicate within host erythrocytes, a process which may lead to life-threatening complications, including severe anemia, splenic rupture, cerebral malaria, respiratory distress, and/or renal failure (41). The intraerythrocytic parasites are somewhat shielded from many cell-mediated and antibody-mediated immune effector mechanisms, and naturally acquired immunity is slow to develop. When the intracellular parasite matures and the host erythrocyte is lysed, the merozoites released are accessible to serum immunoglobulins before they invade new red blood cells (RBCs). While neutralization of free merozoites can occur, plasmodial parasites have also evolved mechanisms to avoid invasion-inhibiting antibodies. There are several alternate invasion pathways that depend on complex interactions between sets of merozoite proteins and host erythrocyte receptors (2, 4, 19, 27, 42, 52). This redundancy can allow invasion to occur even if one receptor-ligand interaction is blocked. In addition, merozoite-neutralizing antibodies are often strain specific due to a significant degree of polymorphism in many merozoite surface antigens (2, 4, 31).

It is also well established that different species and/or strains of malaria parasites preferentially invade erythrocytes of various ages. One of the two major human malarial parasites, Plasmodium vivax, is reticulocyte restricted, while Plasmodium falciparum invades normocytes as well as reticulocytes (2). Host cell tropism may be mediated largely by the differential expression and/or utilization of certain merozoite proteins during the invasion process. In fact, several plasmodial reticulocyte-binding and normocyte-binding proteins have been identified (2, 4). However, malaria parasites also vary in the ability to sequester in certain host tissues (3). The degree to which merozoites are accessible to reticulocytes in the spleen or bone marrow during acute malaria may also contribute to the preferential invasion of subpopulations of host erythrocytes.

Merozoite surface protein-1 (MSP-1), a 195-kDa protein essential for parasite survival, is believed to be one of the key parasite proteins involved in merozoite invasion of host erythrocytes (4, 31). MSP-1 and its processed fragments are part of a high-molecular-weight complex anchored to the parasite surface by a glycolipid moiety (33). The 19-kDa C-terminal fragment of MSP-1 is characterized by the presence of two conserved epidermal growth factor (EGF)-like domains (8). An array of evidence from in vivo and in vitro studies suggests that antibodies directed against these EGF-like domains are protective, presumably due to their ability to inhibit merozoite invasion of erythrocytes (7, 15, 16, 22, 28, 31, 37, 43, 49).

MSP-8 is another glycolipid-anchored surface protein that also contains two C-terminal EGF-like domains (10). Humans naturally infected with P. falciparum produce antibodies against multiple epitopes of P. falciparum MSP-8 (PfMSP-8) (6), and immunization of mice with recombinant Plasmodium yoelii MSP-8 (rPyMSP-8) confers protection against rodent malaria (10). The specific function(s) of MSP-8 in blood-stage parasites is not fully understood. However, allelic replacement experiments indicate that the EGF-like domains of MSP-1 can be functionally replaced with those of MSP-8 (20), suggesting that there is a redundant role for these protein domains in merozoite attachment to and/or invasion of RBCs.

In studies of plasmodial antigens and pathways of erythrocyte invasion, conclusions have been drawn mainly based on the ability of merozoite-specific antibodies to block P. falciparum invasion of mature RBCs in vitro. Supporting in vivo studies utilizing rodent and/or simian models have not routinely distinguished the ability of antibodies to block the invasion of normocytes from the ability of antibodies to block the invasion of reticulocytes. In the present study, the ability of PyMSP-8-immunized mice to suppress infection of mature RBCs and reticulocytes was evaluated by using the 17XL and 17X strains of P. yoelii, respectively. The nature of the protective response induced by PyMSP-8 immunization was investigated in studies of immunologically intact and B-cell-deficient mice immunized with either refolded or denatured PyMSP-8. Finally, in an effort to obtain information on mechanisms underlying an alteration of host cell tropism in immunized animals, changes in gene expression in parasites under immune pressure were measured using P. yoelii DNA microarrays.


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MATERIALS AND METHODS
 
Mice and parasites. Male BALB/cByJ mice and B-cell-deficient JHD mice (13) with a BALB/c background that were 5 to 6 weeks old were purchased from The Jackson Laboratory (Bar Harbor, Maine) and Taconic Farms Inc. (Germantown, NY), respectively. All animals were housed in the Animal Care Facility of Drexel University College of Medicine under specific-pathogen-free conditions. The lethal 17XL and nonlethal 17X strains of P. yoelii were originally obtained from William P. Weidanz (University of Wisconsin, Madison).

Production of rPyMSP-8. The expression and purification of full-length rPyMSP-8 from P. yoelii 17XL using the pET-15b expression vector and Escherichia coli BL21(DE3)(pLysS) as the host strain (Novagen, Madison, WI) have been described previously (10). rPyMSP-8 was purified by nickel chelate affinity chromatography under denaturing conditions and was refolded by gradual removal of guanidine-HCl by dialysis in the presence of reduced and oxidized glutathione (51). Alternatively, the eluted rPyMSP-8 was reduced by treatment with 25 mM dithiothreitol overnight at 4°C and for 1 h at 37°C and was alkylated by treatment with 125 mM iodoacetic acid for 1 h at 37°C.

Refolded rPyMSP-8 and reduced and alkylated rPyMSP-8 (R/A rPyMSP-8) were dialyzed into 25 mM Tris-HCl (pH 8.0) and 100 mM NaCl. Protein concentrations were determined using the bicinchoninic protein assay (Pierce Chemical Company, Rockford, IL), and purity was assessed by Coomassie blue staining following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Refolded rPyMSP-8 migrated as a predominant band at ~54 kDa in the presence of 2-mercaptoethanol (Fig. 1A, lane 2) and as a faster-migrating doublet in the absence of 2-mercaptoethanol (Fig. 1B, lane 2). Higher-molecular-weight aggregates of refolded rPyMSP-8 run under nonreducing conditions were minimal. As expected, R/A rPyMSP-8 migrated as a single band at ~55 kDa in the presence or absence of 2-mercaptoethanol (Fig. 1A and 1B, lane 1).



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  		FIG. 1. Purified recombinant PyMSP-8. Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel run under reducing (A) or nonreducing (B) conditions containing R/A rPyMSP-8 (3 µg) (lane 1) and refolded rPyMSP-8 (3 µg) (lane 2). The positions of molecular mass markers (lane M) are indicated on the left.

Immunizations and experimental infections. Groups of four or five BALB/cByJ or B-cell-deficient JHD mice were immunized subcutaneously with 5 µg or 25 µg of purified rPyMSP-8 (refolded or reduced and alkylated) along with 25 µg of Quil A as an adjuvant (Accurate Chemical and Scientific Corporation, Westbury, NY). Control animals were immunized with Quil A in saline or with saline alone. Mice received three immunizations with the same doses of antigen and adjuvant at 3-week intervals. Two weeks following the last immunization, blood-stage infections were initiated by intraperitoneal injection of 1 x 105 P. yoelii-parasitized erythrocytes. The resulting parasitemias were monitored by using thin tail blood smears stained with Giemsa stain. Polychromatophilic RBCs were counted as reticulocytes. Animals infected with the lethal P. yoelii 17XL strain were removed from the study when the parasitemia exceeded 50% or when they became moribund. In lethal P. yoelii 17XL infections, the significance of differences in the number of surviving animals between groups was determined by the Mantel-Haenszel log rank test (GraphPad Prism 4.0; GraphPad Software Inc., San Diego, CA). In nonlethal P. yoelii 17X infections, the significance of differences in mean peak parasitemia between groups was calculated by analysis of variance.

ELISA. Approximately 2 to 3 days prior to P. yoelii challenge infection, a small volume of serum was collected from rPyMSP-8-immunized and adjuvant control mice. The levels of antibodies present in prechallenge sera that were reactive with refolded and R/A rPyMSP-8 were determined by an enzyme-linked immunosorbent assay (ELISA). Equivalent binding of refolded and R/A rPyMSP-8 to ELISA wells was monitored by reactivity with INDIA HisProbe-horseradish peroxidase (Pierce Chemical Company), which binds to polyhistidine-tagged fusion proteins. Antigen-coated wells were washed and blocked for 1 h with TBS (25 mM Tris-HCl, pH 8, 150 mM NaCl) containing 5% nonfat dry milk. The reactivity of each serum, serially diluted (1:1,000 to 1:32,000) in TBS-0.1% Tween 20 containing 1% bovine serum albumin, was determined with bound antibodies detected using horseradish peroxidase-conjugated rabbit antibody specific for mouse immunoglobulin G (IgG) (Zymed Laboratories, South San Francisco, CA). For each dilution, the mean absorbance of sera from adjuvant control mice (n = 5) was subtracted as the background. The statistical significance of differences in antibody responses between groups was calculated by analysis of variance.

Sequencing MSP-8 from P. yoelii 17X. Genomic DNA was purified from P. yoelii 17X blood-stage parasites as previously described (11). Based on the sequence of PyMSP-8 of 17XL (accession no. AY005132) (10), the complete coding region of PyMSP-8 of 17X was PCR amplified using oligonucleotide primers 5'-CCTTAATTCTAACAACCCGCA-3' (nucleotides 115 to 135) and 5'-AACTTCATAAGATAT GTGCCA-3' (nucleotides 1621 to 1641) and Platinum High Fidelity Taq DNA polymerase (Invitrogen Corporation, Carlsbad, CA) and sequenced.

P. yoelii DNA microarray analysis. P. yoelii DNA microarrays were produced in the Molecular Genomics Core Facility, Drexel University College of Medicine, under the direction of L.W.B. Each spotted array contained 65-base oligonucleotides representing ~6,700 open reading frames predicted from the analysis of the P. yoelii genomic sequence (12). Details of the design and production of the P. yoelii DNA microarrays will be described elsewhere (Bergman, unpublished data).

P. yoelii 17XL blood-stage RNA was isolated from rPyMSP-8-immunized mice (day 13, n = 4) and adjuvant control mice (day 8, n = 4) when the average levels of ascending parasitemia were approximately 15% to 20%. Similarly, P. yoelii 17X blood-stage RNA was isolated from naïve mice (day 13, n = 4) infected with 1 x 105 P. yoelii 17X-parasitized RBCs. It should be noted that the replication of P. yoelii blood-stage parasites is asynchronous and that the distributions of asexual developmental stages in Giemsa-stained thin blood films of sampled animals were comparable. Following saponin lysis of erythrocytes and leukocytes, P. yoelii parasites were resuspended in the TRIzol reagent (Invitrogen), and total parasite RNA was extracted, precipitated, and purified using an RNeasy RNA isolation kit (QIAGEN, Inc., Valencia, CA). Equal quantities of RNA from each group (n = 4) were pooled for gene expression studies.

The gene expression patterns in P. yoelii 17XL parasites isolated from rPyMSP-8-immunized mice were compared to the gene expression patterns in P. yoelii 17XL parasites isolated from adjuvant control animals. In a control set of arrays, gene expression in P. yoelii 17X parasites (primarily in reticulocytes) was compared to gene expression in P. yoelii 17XL parasites (primarily in mature RBCs). To prepare each probe, 10 µg of P. yoelii RNA was reverse transcribed into cDNA in the presence of aminoallyl-dUTP (FairPlay microarray labeling kit; Stratagene, La Jolla, CA). The cDNA was then fluorescently labeled by reaction with monofunctional, normal N-hydroxysuccinimide-activated Cy3 or Cy5 dye (Amersham Biosciences Inc., Piscataway, NJ). The Cy dye-labeled cDNA was purified, and the yield and specific activity of each probe were determined by absorption spectroscopy. Pairs of Cy3- and Cy5-labeled probes were pooled and hybridized to the P. yoelii microarrays for 14 to 16 h at 60°C in a 60-µl mixture. Following hybridization and washing, slides were scanned using a GenePix 4000A microarray laser scanner (Axon Instruments Inc., Union City, CA), and the fluorescence intensity of each DNA feature was determined at 532 nm (Cy3) and at 635 nm (Cy5). Data for each gene were obtained from replicate features (n = 2). On a second set of arrays the assignments of Cy3 and Cy5 for labeling of the paired cDNA probes were reversed (standard dye flip). Gene expression data were acquired and analyzed using the GenePixPro 5.0 and Acuity 3.0 Microarray Informatics software (Axon Instruments). Genes of interest were identified as the genes whose normalized expression was more than twofold greater than that of negative control features (15 randomized oligonucleotides) with at least a twofold change (positive or negative) in gene expression for two data points and at least a 1.5-fold change for all four data points.

Quantitative real-time PCR. Real-time PCR was used to validate microarray expression data for 13 selected genes. Customized primer sets (see Table S1 in the supplemental material) were generated for each P. yoelii antigen gene of interest, as well as three control ribosomal protein genes, using the Primer3 software (primer3_www.cgi v 0.2; Whitehead Institute for Biomedical Research) (50). All reactions were run in duplicate using an ABI Prism 7700 sequencing detection system, and data were analyzed using the Applied Biosystems Sequence Detector (v.1.7) program. Serial dilutions of input cDNA (6 ng/well to 300 pg/well) were used to generate a standard curve for each target gene and the 60S ribosomal protein L23 as the endogenous reference. The relative expression level of each target gene normalized to the endogenous control was determined based on the threshold cycle of product detection. The fold change in the expression of each gene in P. yoelii 17XL parasites from rPyMSP-8-immunized mice was calculated relative to the expression in P. yoelii 17XL parasites from adjuvant control mice.

Nucleotide sequence accession number. The complete sequence of MSP-8 from P. yoelii 17X has been deposited in the GenBank database under accession number AY864847.


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RESULTS
 
rPyMSP-8-induced protection targets parasites that invade mature red blood cells but not reticulocytes. Lethal P. yoelii 17XL parasites have the capacity to invade both normocytes and reticulocytes. In contrast, the nonlethal 17X strain of P. yoelii preferentially invades reticulocytes. Due to its location on the merozoite surface, it is likely that PyMSP-8 plays a role in erythrocyte invasion. To compare the efficacies of rPyMSP-8 immunization against these lethal and nonlethal parasites, mice were immunized with 5 µg of refolded rPyMSP-8 formulated with Quil A as an adjuvant, and the protective efficacy was evaluated following a challenge infection.

As shown in Fig. 2A, all mice in the adjuvant control group developed fulminate, lethal malaria 8 to 10 days following challenge with P. yoelii 17XL. In contrast, all animals immunized with rPyMSP-8 plus Quil A survived the challenge infection. In this immunized group, peak parasitemia occurred between days 12 and 16, and the mean level was 20.5% ± 10.1%. As shown in Fig. 2B, P. yoelii 17X parasitemia was somewhat reduced in rPyMSP-8-immunized mice, which had a mean peak level of parasitemia of 27.3% ± 9.6%, compared to the mean peak levels of parasitemia of 42.1% ± 15.2% and 43.1% ± 18.2% for the Quil A and saline control groups, respectively. The reduction in parasitemia, however, was not statistically significant (P > 0.05). Increasing the dose of rPyMSP-8 from 5 µg to 25 µg per immunization did not improve the protective efficacy (data not shown). These data indicate that rPyMSP-8 immunization induced better protection against lethal P. yoelii 17XL parasites than against nonlethal, reticulocyte-restricted P. yoelii 17X parasites.



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  		FIG. 2. PyMSP-8-induced protection against lethal and nonlethal strains of P. yoelii. Groups of BALB/cByJ mice (four or five mice/group) were immunized with 5 µg of purified rPyMSP-8 formulated with Quil A as an adjuvant ({blacksquare}), with Quil A alone ({diamond}), or with saline alone ({circ}). Approximately 10 to 14 days following the third immunization, the mice were challenged with 1 x 105 P. yoelii 17XL-parasitized erythrocytes (A) or P. yoelii 17X-parasitized erythrocytes (B). The resulting parasitemia was monitored by enumerating parasitized RBCs in thin tail blood smears stained with Giemsa stain. The numbers followed by D indicate the number of deceased animals at each time.

Protection induced by rPyMSP-8 immunization is B cell dependent. To investigate the underlying mechanism of immunity, B-cell-deficient JHD mice or immunologically intact BALB/cByJ mice were concurrently immunized with rPyMSP-8. As shown in Fig. 3A, immunization of immunologically intact mice with rPyMSP-8 induced solid protection against P. yoelii 17XL challenge infection, and the peak level of parasitemia was 12.3% ± 10.5% on day 12 of infection. All adjuvant and saline control mice developed unremitting parasitemia by day 8 postchallenge. B-cell-deficient JHD mice immunized with rPyMSP-8 failed to control P. yoelii parasitemia and succumbed to infection by day 9 postchallenge (Fig. 3B). Thus, protection against lethal P. yoelii blood-stage malaria induced by rPyMSP-8 immunization was predominantly, if not completely, B cell dependent.



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  		FIG. 3. Protection induced by rPyMSP-8 immunization is B cell dependent. Groups of BALB/cByJ mice (A) or B-cell-deficient JHD mice (B) (n = 5) were immunized with 5 µg of purified rPyMSP-8 formulated with Quil A as an adjuvant ({blacksquare}), with Quil A alone ({diamond}), or with saline alone ({circ}). Approximately 10 to 14 days following the third immunization, the mice were challenged with 1 x 105 P. yoelii 17XL-parasitized erythrocytes, and parasitemia was monitored as described in the legend to Fig. 2.

Protective antibodies recognize conformation-dependent B-cell epitopes of rPyMSP-8. In the mature protein, PyMSP-8 is predicted to contain 16 cysteine residues, 12 of which are associated with the two C-terminal EGF-like domains. To determine if the protective anti-PyMSP-8 antibody response was directed against disulfide-dependent epitopes, mice were immunized as described above with refolded rPyMSP-8 or R/A rPyMSP-8. Two weeks after the final immunization, prechallenge serum samples were collected. The vaccine efficacy was measured following challenge infection with P. yoelii 17XL.

As shown in Fig. 4A, immunization with R/A rPyMSP-8 or refolded rPyMSP-8 induced a high level of PyMSP-8-specific antibodies. There was no significant difference between the IgG levels induced by immunization with R/A PyMSP-8 and the IgG levels induced by immunization with refolded rPyMSP-8, as measured against the same antigen used for immunization. A high percentage of the antibodies induced by immunization with R/A rPyMSP-8 were also cross-reactive with refolded rPyMSP-8 (Fig. 4A). A significantly lower percentage of the antibodies induced by immunization with refolded rPyMSP-8 immunization also bound to R/A rPyMSP-8 (P < 0.05). These data indicate that immunization with refolded rPyMSP-8 elicits production of antibodies that largely recognize disulfide-dependent epitopes, while immunization with R/A rPyMSP-8 induces a similar level of antibodies that mainly bind to linear epitopes.



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  		FIG. 4. Protective antibodies recognize conformational epitopes of rPyMSP-8. (A) Groups of BALB/cByJ mice (four or five mice/group) were immunized with R/A rPyMSP-8 (left panel) or refolded (RF) rPyMSP-8 (right panel) formulated with Quil A as an adjuvant or with Quil A alone. The levels of IgG antibodies present in prechallenge sera that recognized R/A rPyMSP-8 (open bars) or refolded rPyMSP-8 (solid bars) as determined by an ELISA were compared. The results are expressed as the background-corrected means and standard deviations for optical density at 405 nm (O.D. 405) determined for prechallenge sera diluted 1:16,000 and 1:32,000. (B) The same groups of BALB/cByJ mice immunized with R/A rPyMSP-8 ({square}), refolded rPyMSP-8 ({blacksquare}), or adjuvant alone ({diamond}) were challenged with 1 x 105 P. yoelii 17XL-parasitized erythrocytes, and parasitemia was monitored as described in the legend to Fig. 2.

As shown in Fig. 4B, all animals immunized with refolded rPyMSP-8 survived an otherwise lethal P. yoelii infection and cleared blood-stage parasites by day 24 postchallenge. In contrast, all animals immunized with R/A rPyMSP-8, as well as adjuvant controls, succumbed to infection by day 11. These data indicate that antibodies induced by rPyMSP-8 immunization that mediate protection recognize conformational, reduction-sensitive epitopes.

Protective B-cell epitopes of PyMSP-8 are not polymorphic. To evaluate polymorphism associated with protective epitopes of PyMSP-8, the Pymsp-8 gene from P. yoelii 17X parasites was cloned and sequenced and compared to that of P. yoelii 17XL parasites. Sequence analysis revealed 100% amino acid identity between the MSP-8 sequences of P. yoelii 17X and 17XL (sequences deposited in the GenBank database). As such, the strain-specific protection induced by immunization with rPyMSP-8 was not due to variation in protective T- or B-cell epitopes.

Changes in gene expression patterns of P. yoelii 17XL parasites from rPyMSP-8-immunized mice. At peak parasitemia in rPyMSP-8-immunized mice, the majority of the P. yoelii 17XL-infected cells (75% to 90%) were reticulocytes. This shift in infection from normocytes to reticulocytes occurred despite the presence of a large number of mature RBCs in circulation (Fig. 5). When P. yoelii-parasitized reticulocytes from rPyMSP-8-immunized mice were transferred into a naïve animal, a fulminant, lethal infection of mature RBCs resulted (data not shown). These observations suggested that changes in gene expression leading to a shift in host cell preference could contribute to the ability of P. yoelii parasites to avoid neutralization by PyMSP-8-specific antibodies.



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  		FIG. 5. P. yoelii 17XL parasites preferentially invade reticulocytes in rPyMSP-8-immunized mice: Giemsa-stained thin tail blood smears from P. yoelii 17XL-infected control mice (A) (day 6) or rPyMSP-8-immunized mice (B) (day 12) during the period of ascending parasitemia.

Changes in P. yoelii 17XL gene expression associated with rPyMSP-8 immunization were evaluated using P. yoelii genomic DNA microarrays. When the average levels of parasitemia were 15% to 20%, total P. yoelii RNA was isolated from rPyMSP-8-immunized mice as well as adjuvant control mice challenged with lethal P. yoelii 17XL or from naïve mice infected with nonlethal P. yoelii 17X. As shown in Table 1, reticulocytes were preferentially parasitized in naïve mice infected with P. yoelii 17X and rPyMSP-8-immunized mice infected with P. yoelii 17XL. Parasites were found almost exclusively in normocytes in control mice infected with P. yoelii 17XL.


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  		TABLE 1. Host cell preference of P. yoelii blood-stage parasites from rPyMSP-8-immunized and control mice

Using P. yoelii genomic DNA microarrays, gene expression in P. yoelii 17XL parasites from rPyMSP-8-immunized animals was compared to gene expression in P. yoelii 17XL parasites from adjuvant control mice. The expression of the vast majority of genes, including those whose expression is developmentally regulated, remained unchanged. These data indicate that the distributions of ring-, trophozoite-, and schizont-stage parasites in the samples were equivalent. As indicated in Fig. 6, only 398 genes were consistently identified that were differentially expressed (269 upregulated genes and 129 downregulated genes) in P. yoelii 17XL parasites obtained from rPyMSP-8-immunized mice compared to controls. Based on P. yoelii genome database information (TIGR.org and PlasmoDB.org), 233 of these genes were predicted to contain a signal sequence and/or at least one transmembrane domain and are therefore likely to be secreted or membrane associated; 243 genes are predicted to have P. falciparum orthologs. Of the 398 differentially expressed genes, 86 were similarly regulated in reticulocyte-associated P. yoelii 17X parasites compared to the control, normocyte-associated P. yoelii 17XL parasites and may be related directly to parasite invasion of and/or growth within reticulocytes (see Table S2 in the supplemental material).



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  		FIG. 6. Summary of changes in gene expression in P. yoelii 17XL parasites from rPyMSP-8-immunized mice. The gene expression patterns in P. yoelii 17XL parasites from control mice and mice immunized with rPyMSP-8 were compared using P. yoelii DNA microarrays. The histograms show the number of differentially expressed genes, as follows: Total, genes upregulated (open bar) or downregulated (shaded bar) in P. yoelii 17XL parasites from rPyMSP-8-immunized mice; SS/TM, subset of genes predicted to contain signal sequences (SS) or transmembrane domains (TM); Pfal, subset of genes predicted to have P. falciparum orthologues; TR, differentially expressed genes associated with a shift in host cell tropism (see Table S2 in the supplemental material); IR, differentially expressed genes associated with rPyMSP-8 immunization (see Table S3 in the supplemental material).

Expression data for the subset of genes of interest are shown in Table 2 and in Tables S2 and S3 in the supplemental material. These data include the fold change in gene expression (means ± standard deviations; n = 4) as determined by microarray analysis, as well as confirmatory real-time PCR data. It was particularly interesting that the expression of PyMSP-8 was approximately twofold lower in P. yoelii 17XL parasites from rPyMSP-8-immunized animals. In contrast, the expression of MSP-1, MSP-4/5, and MSP-7 was somewhat increased. There was little or no change in the expression of other merozoite surface proteins, including apical membrane antigen-1, MAEBL, MSP-9, and MSP-10. Modest increases in the expression of rhoptry-associated protein-1 (RAP1), RAP2, and several members of the 235-kDa rhoptry protein family were noted. The significant changes in expression of ~30 members of the large yir gene family, as well as nine members of a second multigene family (Pyst-a family) predicted to encode erythrocyte membrane-associated proteins were unexpected. Four of the yir genes and three of the Pyst-a genes were similarly regulated in P. yoelii 17X parasites and P. yoelii 17XL controls (see Table S2 in the supplemental material). These data suggest that changes in the expression of merozoite surface antigens, as well as parasite proteins associated with the erythrocyte membrane, may influence host cell tropism and allow blood-stage parasites to evade an otherwise protective, MSP-8-specific immune response.


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  		TABLE 2. Changes in gene expression in P. yoelii 17XL parasites from rPyMSP-8-immunized mice


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DISCUSSION
 
Merozoite surface antigens are of significant interest as potential vaccine candidates due to their role in parasite invasion of erythrocytes and accessibility to host antibodies (31). MSP-8 is one of five glycolipid-anchored, merozoite surface proteins characterized by the presence of one or two EGF-like domains (5, 6, 8, 10, 38, 39). In this paper, we report that immunization with rPyMSP-8 protected mice against otherwise lethal infection with P. yoelii 17XL. Such protection was largely, if not solely, antibody dependent, as B-cell-deficient JHD mice immunized with rPyMSP-8 could not suppress blood-stage parasitemia. We further showed that immunization of immunologically intact mice with R/A rPyMSP-8 induced a strong antibody response against linear determinants that failed to protect against P. yoelii malaria. These data support the conclusion that antibodies that recognize disulfide bond-dependent epitopes of PyMSP-8 are required for protection. These results are not surprising considering the importance of protective antibodies that recognize conformational epitopes of MSP-1 (7, 15, 16, 22, 28, 31, 37, 43, 49), AMA-1 (14, 21, 30), and EBA-175 (1, 52), which are leading blood-stage vaccine candidate antigens.

Although MSP-1 appears to be essential for the growth of blood-stage malarial parasites, Drew et al. (20) showed that the EGF-like domains of P. falciparum MSP-1 can be replaced by the corresponding domains of Plasmodium berghei MSP-8. Since the EGF-like domains of MSP-1 and MSP-8 differ significantly in the primary amino acid sequence, these data suggest that the observed functional redundancy may be related to the conservation of protein structure. As the EGF-like domains of PfMSP-1 and PfMSP-8 do not appear to be serologically cross-reactive (6), our data suggest that immunization with combined formulations of MSP-142 and MSP-8 may be necessary to adequately inhibit their function and achieve an acceptable level of vaccine efficacy. From this perspective, the limited sequence diversity in the msp-8 gene of several P. falciparum strains is advantageous (6).

The potential redundancy in function of the EGF-like domains of MSP-1 and MSP-8 highlights one of the major challenges for merozoite antigen-based vaccines. Merozoites can invade erythrocytes by alternate, sometimes nonoverlapping pathways that involve distinct receptor-ligand interactions (2, 4, 19, 27, 42, 52). This is further complicated in the case of P. falciparum, which has the capacity to invade reticulocytes as well as normocytes. As shown here, immunization with rPyMSP-8 protects against infection with lethal P. yoelii 17XL, which can invade normocytes as well as reticulocytes (23, 54). In contrast, immunization with rPyMSP-8 does not significantly alter the course of infection with nonlethal P. yoelii 17X parasites, which exhibit a predilection for reticulocytes. This strain-specific protection is not related to antigen polymorphism, as no differences were identified in the amino acid sequences of PyMSP-8 from the 17X and 17XL strains of P. yoelii.

Closer examination of the P. yoelii 17XL infection in rPyMSP-8-immunized mice revealed that the parasitemia that developed 10 to 18 days postchallenge was restricted to reticulocytes despite the presence of a large number of mature RBCs in circulation. A similar finding has been reported for mice immunized with a 235-kDa rhoptry protein of P. yoelii 17XL (25, 32). Similar to human malaria parasites, strains of P. yoelii appear to use multiple erythrocyte receptors for invasion. The Duffy antigen/receptor for chemokines is one receptor that is preferentially utilized by P. yoelii during invasion of mature RBCs, while as-yet-undefined receptors appear to be used for invasion of reticulocytes (53). The members of the Py235 family of rhoptry proteins have been shown to bind to the surface of uninfected erythrocytes (44). It has been suggested that the differential expression of these rhoptry proteins by P. yoelii blood-stage parasites may contribute to the selection of host cells for invasion (26, 36, 48). As it appears that anti-PyMSP-8 antibodies block the infection of mature RBCs but not reticulocytes (Fig. 7A and 7B), it is possible that MSP-8 is not required for the invasion of reticulocytes. However, our preliminary findings indicate that rPyMSP-8 does bind to the surface of normocytes, as well as reticulocytes (Shi and Burns, unpublished observations). Perez-Leal et al. (47) have also reported that P. vivax blood-stage parasites, which are known to infect only reticulocytes, do express P. vivax MSP-8.



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  		FIG. 7. Potential mechanisms for the alteration of P. yoelii 17XL host erythrocyte tropism in rPyMSP-8-immunized mice. Merozoite invasion pathways involving normocytes and reticulocytes are shown in red and blue, respectively. (A) In a naïve host, P. yoelii 17XL merozoites preferentially invade normocytes. (B) In the presence of anti-PyMSP8 antibodies, merozoite invasion of normocytes is blocked, and reticulocytes are preferentially invaded. (C) Changes in gene expression result in an increase in the expression of merozoite proteins that preferentially bind reticulocytes over normocytes. (D) Changes in the expression of yir and Pyst-a gene products on the surface of infected erythrocytes promote the localization of schizont-infected erythrocytes to the reticulocyte-rich region of the spleen. In this compartment, exposure of released merozoites to neutralizing antibodies is reduced, and the invasion of reticulocytes is highly efficient.

We considered the possibility that P. yoelii 17XL parasites might modulate the expression of PyMSP-8 and/or other merozoite surface proteins in order to escape neutralization by anti-PyMSP-8 antibodies (Fig. 7A and 7C). Our analysis of P. yoelii gene expression indicated that the transcription of Pymsp-8 was reduced approximately twofold in P. yoelii 17XL parasites obtained from rPyMSP-8-immunized mice compared to controls. Of interest, a ~twofold increase in the transcription of Pymsp-1 and Pymsp-7 was also noted. These reciprocal changes, while modest, are of interest considering the potential overlap in functions of the EGF-like domains of MSP-1 and MSP-8 and the coexpression of MSP-1 and MSP-7 as part of a high-molecular-weight surface complex (40, 46). However, this shift in msp-1, msp-7, and msp-8 expression did not appear to depend on the presence of anti-PyMSP-8 antibodies as similar changes were observed in P. yoelii 17X parasites from naïve animals compared to P. yoelii 17XL controls. Also notable was the increase in expression of several rhoptry proteins, including the putative P. yoelii orthologs of P. falciparum RAP1 and RAP2 and a subset of the Py235 gene family. For the most part, the expression of this set of rhoptry protein genes was similarly upregulated in P. yoelii 17X parasites compared to P. yoelii 17XL controls. These changes likely reflect the role of certain rhoptry proteins in virulence and/or in the invasion of reticulocytes, as has been previously reported (26, 36, 48).

Our genome-wide analysis of changes in gene expression in P. yoelii 17XL parasites from rPyMSP-8-immunized mice had additional unexpected results. In P. yoelii 17XL parasites from rPyMSP-8-immunized mice there was increased expression of a subset of genes belonging to the yir and Pyst-a multigene families. The ~800 members of the yir gene family are predicted to encode antigenically variable proteins that are expressed on the surface of infected erythrocytes, and similar large multigene families have been identified in P. vivax (vir), Plasmodium knowlesi (kir), Plasmodium chabaudi (cir), and P. berghei (bir) (12, 17, 24, 34, 35). The Pyst-a multigene family contains approximately 140 members, and the products of many of these members are predicted to contain signal sequences and/or transmembrane domains (12). A multigene family related to the P. yoelii Pyst-a genes has been identified in P. chabaudi, and the genes encode 90- to 100-kDa antigens that are also expressed at the surface of infected erythrocytes and are the targets of protective immune responses (24, 55, 57).

How changes in the expression of parasite proteins at the erythrocyte surface could influence host cell preference and/or enable P. yoelii parasites to avoid PyMSP-8-specific, neutralizing antibodies is an interesting question. Bucsu et al. (9) observed changes in the expression of parasite antigens on the surface of infected erythrocytes with recrudescent P. chabaudi parasites obtained from mice protected by immunization with recombinant AMA-1. Unlike P. falciparum and to some degree P. chabaudi, P. yoelii parasites are not known for their ability to cytoadhere to vascular endothelium. However, the abilities of P. yoelii parasites to leave the peripheral circulation and enter the erythropoietic regions of the spleen are different for the 17X and 17XL strains of P. yoelii (56). One suggestion is that this may be regulated in part by the differential production of cytokines during infection (45). We propose an alternative (Fig. 7D). The differential expression of yir- and/or Pyst-a-encoded proteins on the surface of infected erythrocytes could enable parasitized cells to pass into and/or sequester within the red pulp of the spleen. Intrasplenic infection of reticulocytes could occur more readily as P. yoelii merozoites released in the spleen may not be exposed to sufficient levels of protective antibodies. A similar role for the vir genes of P. vivax has recently been postulated (18).

Our studies suggest that it is still necessary for rPyMSP-8-immunized animals to mount an immune response to other parasite proteins in order to fully suppress blood-stage malaria. Similar conclusions have been drawn from studies of PyMSP-1 involving both active and passive immunization experiments (15, 29). We predict that combined formulations of PyMSP-1, PyMSP-8, and one or more rhoptry proteins are necessary to fully neutralize merozoites. In addition, it may be necessary to concurrently target parasite proteins expressed at the erythrocyte surface to prevent sequestration of parasitized erythrocytes in host tissues in order to increase exposure to merozoite-neutralizing antibodies. This may present a significant challenge for the development of P. falciparum and P. vivax blood-stage malaria vaccines as the target antigens at the infected erythrocyte membrane may include members of large, antigenically variable protein families. Using the P. yoelii model, it should be possible to determine if the more conserved domains of the yir- and/or Pyst-a-encoded antigens can be targeted for immunization and used to improve the efficacy of a multiantigen blood-stage malaria vaccine.


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ACKNOWLEDGMENTS
 
This work was supported by NIH-NIAID grants R01AI35661 (to J.M.B) and R21AI53808 (to L.W.B.).


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FOOTNOTES
 
* Corresponding author. Mailing address: Drexel University College of Medicine, Department of Microbiology and Immunology, 2900 Queen Lane, Philadelphia, PA 19129. Phone: (215) 991-8490. Fax: (215) 848-2271. E-mail: jburns{at}drexelmed.edu. Back

{dagger} Supplemental material for this article may be found at http://iai.asm.org/. Back

Editor: W. A. Petri, Jr.


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  • Petritus, P. M., Burns, J. M. Jr. (2008). Suppression of Lethal Plasmodium yoelii Malaria following Protective Immunization Requires Antibody-, IL-4-, and IFN-{gamma}-Dependent Responses Induced by Vaccination and/or Challenge Infection. J. Immunol. 180: 444-453 [Abstract] [Full Text]  
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