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Infection and Immunity, February 2002, p. 820-825, Vol. 70, No. 2
0019-9567/01/$04.00+0     DOI: 10.1128/IAI.70.2.820-825.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Protective Immune Responses to the 42-Kilodalton (kDa) Region of Plasmodium yoelii Merozoite Surface Protein 1 Are Induced by the C-Terminal 19-kDa Region but Not by the Adjacent 33-kDa Region

Niklas Ahlborg,1,{dagger} Irene T. Ling,2 Wendy Howard,1 Anthony A. Holder,2 and Eleanor M. Riley1*

Institute of Cell, Animal and Population Biology, Edinburgh University, Edinburgh EH9 3JT,1 National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom2

Received 18 June 2001/ Returned for modification 17 October 2001/ Accepted 5 November 2001


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Vaccination of mice with the 42-kDa region of Plasmodium yoelii merozoite surface protein 1 (MSP142) or its 19-kDa C-terminal processing product (MSP119) can elicit protective antibody responses in mice. To investigate if the 33-kDa N-terminal fragment (MSP133) of MSP142 also induces protection, the gene segment encoding MSP133 was expressed as a glutathione S-transferase (GST) fusion protein. C57BL/6 and BALB/c mice were immunized with GST-MSP133 and subsequently challenged with the lethal P. yoelii YM blood stage parasite. GST-MSP133 failed to induce protection, and all mice developed patent parasitemia at a level similar to that in naive or control (GST-immunized) mice; mice immunized with GST-MSP119 were protected, as has been shown previously. Specific prechallenge immunoglobulin G (IgG) antibody responses to MSP1 were analyzed by enzyme-linked immunosorbent assay and immunofluorescence. Despite being unprotected, several mice immunized with MSP133 had antibody titers (of all IgG subclasses) that were comparable to or higher than those in mice that were protected following immunization with MSP119. The finding that P. yoelii MSP133 elicits strong but nonprotective antibody responses may have implications for the design of vaccines for humans based on Plasmodium falciparum or Plasmodium vivax MSP142.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The Plasmodium falciparum merozoite surface protein 1 (MSP1) is one of the major surface proteins of the merozoite, and immunization with full-length, native MSP1 protein can protect monkeys from otherwise lethal infection with parasites (29). MSP1 is processed into a complex of polypeptides on the merozoite surface, including N-terminal and central regions of 82, 30, and 38 kDa, as well as the C-terminal region of 42 kDa (18, 24). At the time of invasion of red blood cells (RBC), MSP142 is further processed by proteolytic cleavage into a 33-kDa fragment (MSP133), which is shed with the rest of the complex, and a C-terminal 19-kDa fragment (MSP119) (3). Only the C-terminal MSP119 remains on the merozoite surface and is carried into parasitized RBC (pRBC) (2). In monkeys, immunization with recombinant MSP142 and MSP119 has been shown to elicit various degrees of protection against P. falciparum challenge (6, 11, 19). The importance of the P. falciparum MSP133 region for induction of protective immunity is not known.

MSP1 from the rodent malaria parasite Plasmodium yoelii appears to be processed in a manner similar to that of P. falciparum MSP1 (17), and recombinant P. yoelii MSP119 can elicit protective immunity in mice (8, 15, 21). The immunity induced by MSP119 in mice is antibody dependent (1, 7, 15, 22) and may, to a large extent, be mediated by antibodies that act directly on the parasite (21, 27). A possible mechanism could be interference with the processing of MSP142, resulting in inhibition of merozoite invasion; this has been shown in vitro with antibodies to P. falciparum MSP119 (4). While immunization with recombinant P. yoelii MSP142, which contains both MSP133 and MSP119, protects mice, immunization with recombinant proteins corresponding to N-terminal or central regions of MSP1 has little if any protective effect (34).

P. yoelii MSP133 has not previously been evaluated for its ability to elicit protection. Antibodies to MSP133 could be potentially protective—for example, if they interfere with processing of MSP142, as has been shown for antibodies to P. falciparum MSP119. Alternatively, antibodies to MSP133 may hinder the binding of protective antibodies induced by MSP119 and thus compromise immunity induced by MSP119 alone (14).

In the present study, mice were immunized with P. yoelii MSP133 and challenged with a virulent clone of P. yoelii (YM) to assess the protective capacity of MSP133. Although MSP133 was found to be highly immunogenic and elicited levels of antibodies to native MSP1 that were comparable to the levels induced in mice immunized with MSP119, no protection of MSP133-immunized mice was observed, suggesting that antibody responses to recombinant P. yoelii MSP133 are not protective.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cloning of P. yoelii MSP133 and expression of recombinant GST fusion proteins. In order to express P. yoelii MSP133 as a glutathione S-transferase (GST) fusion protein, genomic DNA from P. yoelii YM was purified using Instagene according to the manufacturer’s instructions (Bio-Rad, Hemel Hempstead, United Kingdom). DNA encoding residues 1394 to 1655 of P. yoelii MSP1 (PyMSP1) (20) was PCR amplified using the sense primer 5'-GCG GAT CCA ATC CGA AGA TGC ACC AGA A-3' and the antisense primer 5'-GCG AAT TCT AGC TGG AAG AAC TAC AGA A-3' (VH BIO, Newcastle, United Kingdom). The PCR product was purified from a 1% agarose gel using Prep-a-gene (Bio-Rad), restricted with BamHI and EcoRI (Promega, Madison, Wis.), and then purified using a High Pure PCR kit (Boehringer GmbH, Mannheim, Germany).

The PCR product was ligated into linearized pGEX-3X (Pharmacia, Uppsala, Sweden), digested with the same restriction enzymes, using T4 DNA ligase (Promega). After transformation of Escherichia coli strain DH5{alpha} with ligated plasmid, positive clones were screened for protein expression after induction with isopropyl-ß-D-thiogalactopyranoside (IPTG) (Sigma, Poole, United Kingdom). The plasmid from a clone expressing a protein of the expected size was purified using Wizard miniprep columns (Promega), and the complete insert was confirmed by DNA sequencing.

For production of recombinant GST fusion proteins, E. coli DH5{alpha} was transformed with plasmids encoding either GST-MSP133, GST-MSP119 (residues 1649 to 1754 [21]), or GST alone. The proteins were produced as described previously (30) with some modifications (1). Briefly, overnight cultures of transformed bacteria were induced with 1 mM IPTG. The cells were harvested and lysed in phosphate-buffered saline (PBS). The lysate was centrifuged and the supernatant was recovered. Recombinant proteins were purified by affinity chromatography on glutathione-agarose (Sigma) using 10 mM reduced glutathione (Sigma) for elution. Free glutathione was removed by dialysis against PBS.

Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that GST-MSP119, GST-MSP133, and GST were predominantly full length and had relative molecular masses of 42, 65, and 26 kDa, respectively. The concentrations of purified proteins were determined by multiplying the absorbance at 280 nm with factors calculated for each protein based on size and content of aromatic amino acids (13).

For production of GST-free MSP119 and MSP133, glutathione-agarose was incubated with supernatants from bacterial culture (21). After extensive washing, the agarose beads were treated with factor Xa (Sigma) for 12 h at 22°C. Factor Xa was removed from the solution containing the GST-free proteins by incubation with p-aminobenzamidine bound to agarose (Sigma). Simultaneous incubation with glutathione-agarose ensured that any contaminating GST was removed. The proteins were dialyzed against PBS and analyzed by SDS-PAGE, and the concentrations were determined as above. Only one band was observed by SDS-PAGE for purified GST-free MSP119 as well as MSP133 and corresponded to relative molecular masses of 20 and 34 kDa, respectively.

P. yoelii parasites. P. yoelii parasites of the lethal strain YM (38), kindly provided by David Walliker, Edinburgh University, were kept frozen at -70°C or by regular passage in mice. Infected mice were given drinking water supplemented with 2.5 g of para-benzoaminoic acid/liter (25).

Immunization and challenge of mice. Female C57BL/6 and BALB/c mice (Harlan, Oxford, United Kingdom) were used at 7 to 10 weeks of age. Groups of five mice were injected intraperitoneally with 40 µg of GST-MSP119, GST-MSP133, or GST in complete Freund’s adjuvant (Sigma). Booster injections were given 3 and 6 weeks later using the same amount of antigen but in incomplete Freund’s adjuvant (Sigma). Prechallenge sera were drawn 2 weeks after the last booster injection. Three weeks after the last immunization, mice were inoculated intravenously with 104 pRBC. Giemsa-stained blood smears were made daily from day 3 of infection onwards. Parasitemia was assessed by counting the proportion of pRBC by light microscopy.

ELISA. GST-free MSP119 or MSP133 was adsorbed to Immunolon 4 plates (Dynatech Laboratories Inc., Billingshurst, United Kingdom) at a concentration of 2 µg/ml in PBS overnight at 8°C. The wells were blocked with 1% bovine serum albumin in PBS for 1 h at room temperature, and then diluted sera were added and incubated for 1 h at 37°C. Bound antibodies were detected by subsequent incubation with goat anti-mouse immunoglobulin G1 (IgG1), IgG2b, or IgG3 (Southern Biotechnology, Birmingham, Ala.) or sheep anti-mouse IgG2a (The Binding Site, Birmingham, United Kingdom) conjugated to horseradish peroxidase. As the substrate, o-phenylenediamine (Sigma) was used. Endpoint serum titers were estimated by analyzing sera at consecutive twofold dilutions, starting at 1:64, using an absorbance value of 0.3 as the cutoff for positive sera. The mean + 2 standard deviations absorbance for normal mouse serum, diluted 1:64, was lower than the cutoff for all antigens analyzed. A high-titer serum pool was included in all assays as an internal standard.

IFAT. For the indirect immunofluorescent antibody test (IFAT), blood was drawn from P. yoelii YM-infected mice into heparinized tubes. The blood was washed and diluted 1:200 in phosphate-saline buffer containing 0.2% glucose. Then 10 µl of cell suspension was added to wells of 15-well multitest slides (ICN Biomedicals Inc., Aurora, Ohio) that were air dried before storage at -20°C. Prior to staining, the slides were fixed with acetone for 2 min and blocked in PBS with 1% bovine serum albumin for 30 min at room temperature. The wells were incubated for 1 h at 37°C with serum in consecutive twofold dilutions in PBS. Specific antibodies were detected by incubation for 1 h at 37°C with fluorescein isothiocyanate-conjugated goat anti-mouse IgG1, IgG2a, IgG2b, or IgG3 (Southern Biotechnology). Between incubations, the slides were washed in PBS. Titers were determined as the reciprocal of the last positive dilution when analyzed by UV microscopy.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Challenge of immunized mice with P. yoelii parasites. BALB/c and C57BL/6 mice were immunized three times with GST-MSP119, GST-MSP133, or GST. Following immunization, the mice were challenged with 104 parasites of the lethal strain P. yoelii YM; naive mice were included for comparison (Fig. 1). In naive mice and GST-immunized control mice, the course of infection was rapid. All mice died between days 7 and 9 with parasitemias above 60%. In BALB/c and C57BL/6 mice immunized with GST-MSP119, protection was observed in four of five mice in each group, with one mouse (BALB/c) being fully protected (i.e., not having any detectable parasitemia). One BALB/c mouse died with fulminant infection at day 10 and thus showed only a minor delay in the course of infection. One C57BL/6 mouse died despite a delayed course of infection (day 15) and a maximal parasitemia of 26%. In mice immunized with GST-MSP133, no protection was observed; all mice died at day 8 or 9, and there was no difference between the two mouse strains.



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  		FIG. 1. Challenge infection with P. yoelii YM of mice immunized with MSP133 or MSP119. Three weeks after the third immunization with GST-MSP133 or -MSP119, groups of four or five C57BL/6 and BALB/c mice were inoculated intravenously with 104 P. yoelii YM. Parasitemia was monitored daily from day 3. Control groups consisted of mice immunized with GST alone and naive mice. Only mice in the GST-MSP119 groups were able to survive the challenge; mice that did not survive are indicated by {dagger}.

 
Antibody responses to GST-MSP1 fusion proteins as measured by ELISA. To evaluate the predictive value of prechallenge IgG antibody levels on protection, sera from all mice immunized with GST-MSP119 and GST-MSP133 were analyzed for IgG subclass reactivity by ELISA and by IFAT. Direct comparison of ELISA IgG titers to MSP119 and MSP133 is not possible because different coating antigens were used in the ELISAs. Nevertheless, the results suggest that BALB/c mice responded with roughly comparable antibody titers to MSP119 and MSP133, although there was some difference in the subclass of the antibodies, with mean titers in the GST-MSP119 group being higher for IgG1 and lower for IgG2a and IgG2b compared with the GST-MSP133 group (Table 1). A similar result was observed for the C57BL/6 mice, although the differences between the groups were more pronounced, with mean IgG1 titers being twice as high for MSP119 as for MSP133 and mean IgG2a titers being threefold higher for MSP133 than for MSP119 (Table 1).


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  		TABLE 1. IgG subclass responses to GST-MSP119 and GST-MSP133 in C57BL/6 and BALB/c mice

 
Antibody responses to GST-MSP1 fusion proteins as measured by IFAT. To compare antibody responses to native MSP1 in mice immunized with GST-MSP119 or GST-MSP133, individual sera from mice in each group were tested by IFAT to determine the endpoint antibody titer and antibody subclass by using acetone-fixed blood from P. yoelii-infected naive mice. Although absolute titers determined by IFAT were lower than those measured by ELISA, IFAT titers correlated well with the ELISA data; the individual IFAT titers for each group of mice are shown in Fig. 2.



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  		FIG. 2. IgG subclass of antibodies to native P. yoelii MSP1 in mice immunized with GST-MSP133 or -MSP119, as measured by IFAT. Sera from C57BL/6 and BALB/c mice immunized three times with either GST-MSP133 or -MSP119 were analyzed by IFAT for reactivity with mouse RBC infected with P. yoelii YM. IFAT reactivity for all four IgG subclasses was measured and is shown as titers, with different bar patterns representing different subclasses. One of the GST-MSP119-immunized BALB/c mice had an IgG1 titer in excess of 100,000 (262,000). IgG3 was only detected in BALB/c mice immunized with GST-MSP133. C indicates mice that cleared their infection; the number in parentheses indicates the day on which the infection was cleared. {dagger} indicates mice that died or were humanely killed, with the day of death in parentheses. Percentages indicate the highest parasitemia detected during the course of infection.

 
Despite the small number of mice included in the study, there was a clear trend towards an association between antibody responses and protection in the GST-MSP119-vaccinated mice, as described previously (1). The fully protected BALB/c mouse had an extremely high IgG1 response in comparison with the other mice, whereas the BALB/c mouse that did not survive had the lowest antibody titer. Also, the C57BL/6 mice with the lowest maximal parasitemias had the highest overall antibody titers in that group. However, the C57BL/6 mouse that died on day 15 with a parasitemia of 26% did not stand out as a low responder in that group.

The mice immunized with GST-MSP133 had, as also observed by ELISA, lower mean titers of IgG1 but higher titers of IgG2a and IgG2b than mice immunized with GST-MSP119. In BALB/c mice, the IgG3 response to GST-MSP133 was also higher than that to GST-MSP119.

Despite the fact that IgG1 responses to GST-MSP133 were generally lower than those to GST-MSP119, some of the nonprotected GST-MSP133-immunized mice had antibody levels that were comparable to or higher than those of several mice that were protected by immunization with GST-MSP119. Two BALB/c mice immunized with GST-MSP133 had IgG1 levels that were comparable to those of three of the protected mice in the GST-MSP119-immunized group. These GST-MSP133-immunized mice also had comparable or higher titers of the other IgG subclasses. Of the C57BL/6 mice immunized with GST-MSP133, one mouse had comparable IgG1 and higher IgG2a and IgG2b titers than two of the mice protected by immunization with GST-MSP119.

It was clear from the IFAT analysis that the specificity of antibodies raised by GST-MSP133 differed from the specificity of antibodies to GST-MSP119. Antisera to GST-MSP119 stained both pigmented schizonts and ring stages, whereas antisera to GST-MSP133 only stained pigmented schizonts.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Immunization of BALB/c mice with P. yoelii MSP142, which comprises both MSP133 and MSP119 polypeptides, elicits protective immune responses (34), but similar levels of protection can be achieved in both BALB/c and C57BL/6 mice by immunization with the MSP119 polypeptide alone (8, 15, 21, 34). The extent to which the MSP133 region contributes to the protective immune response in MSP142-immunized mice is thus rather unclear. The data presented here show that epitopes contained entirely within the MSP133 polypeptide do not mediate protection. This finding is in agreement with the finding that a monoclonal antibody to P. yoelii MSP133 is unable to passively protect mice; this monoclonal antibody binds to an epitope located some distance from the cleavage site within MSP142 (31).

However, another monoclonal antibody, D3, which does passively protect mice against infection, recognizes an epitope in MSP142 that is not present in either MSP119 or MSP133 (31) and may therefore recognize an epitope formed by the physical interaction of the two polypeptides. As the protective capacity of antibodies against P. falciparum MSP119 (PfMSP119) is, at least in part, related to the ability of the antibodies to inhibit proteolytic processing of PfMSP142 (4, 14), antibodies binding to an epitope formed by sequences from both MSP133 and MSP119 and lying close to the cleavage site could inhibit processing and thus contribute to protection. A monoclonal antibody that inhibits P. falciparum invasion may also bind to epitopes formed from both PfMSP133 and MSP119 (37).

In order to examine the protective effect of MSP133, it was expressed as a GST fusion protein (GST-MSP133) and used, in parallel with GST-MSP119, to immunize BALB/c and C57BL/6 mice. The results confirmed the protective effects of vaccination with GST-MSP119; in contrast, no protection was observed in mice immunized with MSP133. Protective immunity in mice immunized with recombinant P. yoelii MSP119 is largely dependent on antibodies; passive transfer of antiserum to MSP119 can confer partial protection on naive recipients, whereas immunoglobulin µ-chain-deficient mice cannot be immunized with MSP119 (7, 15, 22, 23). We therefore investigated whether the lack of protection following immunization with MSP133 was due to an insufficient antibody response.

Although the antibody response to MSP133 was, on average, lower than the response to MSP119, several mice immunized with MSP133 developed an anti-P. yoelii MSP1 antibody response comparable to or higher than the response in mice that were well protected following immunization with GST-MSP119. We therefore conclude that the lack of a sufficient antibody titer is, in itself, not responsible for the failure of GST-MSP133 to induce protection, and we assume that antibodies elicited by recombinant MSP133 are of a specificity that fails to inhibit the growth of parasites.

It has been shown that folding of the MSP119 recombinant protein into the correct three-dimensional conformation is important for its ability to elicit protective antibodies (21). The importance of correct folding of MSP119 is probably related to the high number of cysteine residues, which form multiple disulfide bonds; this is less likely to present problems for expression of MSP133 because it does not contain any disulfide bonds (20). Indeed, antibodies induced by GST-MSP133 reacted strongly with native, parasite-derived antigen in IFAT. It is thus likely that the antibodies to MSP133 are specific for nonprotective epitopes.

A relevant question is whether or not a vaccine based on MSP119 is affected by the inclusion of MSP133. Comparisons of the immunogenicity and protective capacity of P. yoelii MSP119 and MSP142 in BALB/c and A/J mice did not show any difference between the constructs (34). However, it is possible that in some mouse strains which respond less well to MSP119 (33), the addition of MSP133 could provide additional T-cell epitopes to enhance the antibody response to MSP119. T-cell responses are known to contribute to protection induced by MSP119 vaccination and are essential for immune memory (16). The linkage of exogenous T-cell epitopes to MSP119 has been shown to improve protective antibody responses in mouse strains that are poorly protected by immunization with MSP119 alone (1).

The importance of increasing the number of T-helper-cell epitopes in immunogens based on P. falciparum MSP119, intended for use in humans, is not known. Individuals from areas where malaria is endemic who have been immunized by natural infection with P. falciparum are frequently seronegative for antibodies to MSP119 (9, 12, 28, 35), and T-cell proliferative responses to peptides derived from PfMSP119 tend to be infrequent and relatively weak (10, 26, 36). Importantly, however, T-cell proliferative and cytokine responses to recombinant proteins, including the 33-kDa region of PfMSP1, have been associated with protective immunity to malaria in epidemiological studies (26).

Vaccination of nonhuman primates with MSP19 or MSP42 has produced inconsistent and sometimes contradictory results. In one primate immunization study, vaccination with bacterium-derived MSP119 induced only relatively low-titer antibody responses which were not protective (5), whereas in a similar study using a different adjuvant, vaccination with baculovirus-derived PfMSP142 induced strong lymphoproliferative responses and high antibody titers to PfMSP119; vaccine-induced antibodies effectively inhibited parasite growth in vitro, and all immunized animals were protected against challenge infection (6).

In a direct comparison of the efficacy of PfMSP142 and PfMSP119 as immunogens, monkeys immunized with PfMSP119 appeared to be better protected than those immunized with PfMSP142 (19), but the number of animals tested was small and the two antigens were produced in different expression systems (Saccharomyces cerevisiae and E. coli, respectively), which may have contributed to differences in immunogenicity.

In another study, monkeys vaccinated with yeast-derived PfMSP119 were significantly less well protected than monkeys immunized with baculovirus-derived PfMSP142 (32). Inclusion of exogenous T-cell epitopes in the MSP119 construct does not necessarily enhance protection to immediate challenge, although such vaccines seem to induce strong memory responses (11). Further studies are therefore justified to determine the importance of MSP133-derived T helper epitopes in contributing to natural and vaccine-induced immunity to MSP1 in animal models and in human vaccination studies.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This work was funded by a Research Training Grant to N. Ahlborg from the Life Sciences and Technologies Directorate of the European Commission (contract number BMH4-CT97-5037) and by the Wellcome Trust.

We thank S. Haley for technical support and D. Cavanagh and S. Franks for technical advice.


   FOOTNOTES
 
* Corresponding author. Present address: Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel St., London WC1E 7HT, United Kingdom. Phone: 0207 927 2706. Fax: 0207 837 4314. E-mail: e.riley{at}lshtm.ac.uk. Back

Editor: R. N. Moore

{dagger} Present address: Mabtech, SE-131 37 Nacka, Sweden. Back


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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Infection and Immunity, February 2002, p. 820-825, Vol. 70, No. 2
0019-9567/01/$04.00+0     DOI: 10.1128/IAI.70.2.820-825.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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