Skip to main content
Download PDF
  • Original Articles
  • Published:

A Plasmodium vivax Vaccine Candidate Displays Limited Allele Polymorphism, Which Does Not Restrict Recognition by Antibodies

Molecular Medicine volume 5, pages 459–470 (1999)Cite this article

Abstract

Background

The 19 kDa C-terminal region of the merozoite surface protein 1 (MSP119) has been suggested as candidate for part of a subunit vaccine against malaria. A major concern in vaccine development is the polymorphism observed in different plasmodial strains. The present study examined the extension and immunological relevance of the allelic polymorphism of the MSP119 from Plasmodium vivax, a major human malaria parasite.

Materials and Methods

We cloned and sequenced 88 gene fragments representing the MSP119 from 28 Brazilian isolates of P. vivax. Subsequently, we evaluated the reactivity of rabbit polyclonal antibodies, a monoclonal antibody, and a panel of 80 human sera to bacterial and yeast recombinant proteins representing the two allelic forms of P. vivax MSP119 described thus far.

Results

We observed that DNA sequences encoding MSP119 were not as variable as the equivalent region of other species of Plasmodium, being conserved among Brazilian isolates of P. vivax. Also, we found that antibodies are directed mainly to conserved epitopes present in both allelic forms of the protein.

Conclusions

Our findings suggest that the use of MSP119 as part of a subunit vaccine against P. vivax might be greatly facilitated by the limited genetic polymorphism and predominant recognition of conserved epitopes by antibodies.

Introduction

Malaria remains the most prevalent and devastating parasitic disease worldwide, with a yearly estimate of more than 300 million cases due to Plasmodium falciparum and at least 40 million cases due to P. vivax. The population at risk is estimated at billions of individuals living in tropical regions of the world. The disease is most prevalent and deadly in Africa, where P. falciparum is endemic. Although the patterns of disease transmission, infection, and mortality are quite diverse, malaria transmission also occurs in large areas of South and Southeast Asia, Latin America, and Oceania, where malaria caused by P. vivax is widespread.

Although malaria control was successful in many countries in the past, it is now very difficult to eradicate the disease in developing countries, mainly because of the presence of drug-resistant parasites and mosquito vectors. In view of these problems, the development of new strategies for prevention and control of the disease are in great need.

Mass vaccination is considered a feasible objective that in the future may be added to other strategies such as chemotherapy, vector control, etc., for the prevention and control of malaria. One of the most promising vaccine candidates against the erythrocytic forms of malaria is the merozoite surface protein 1 (MSP1, reviewed in ref. 1). This protein is synthesized as a large-molecular-weight precursor (180–230 kDa) during schizogony and is later processed into several smaller fragments corresponding to the major merozoite surface proteins. During the invasion process, a proteolytic cleavage step releases most of the molecule from the merozoite membrane and only a 19 kDa glycerolphosphatidylinositol-anchored fragment of the C-terminus (MSP119) is carried into the invaded red cells (2,3). The biological importance of MSP1 for parasite survival is unknown; however, it is well established that antibodies that recognize its C-terminal region inhibit merozoite invasion in vitro (3–5) and confer passive immunity on naive mice (6,7). Most relevant to malaria control is the fact that immunization with recombinant proteins based on the sequence of MSP119 in human, non-human primate, and rodent species malaria can provide remarkable protection against experimental infection with blood stages of Plasmodium (8–13).

MSP119 of different species consists of two domains, each with six cysteine residues configured as an epidermal growth factor (EGF)-like domain. EGF-like structures are immunogenically very important. Epitopes recognized by protective antibodies are dependent on the conformation provided by the disulfide bonds and the presence of both EGF-like domains (9,14).

Despite the structural conservation, there are several amino acid modifications among MSP119 of different species of malaria. Most relevant for vaccine development is the polymorphism observed in different strains of the same species. A series of amino acid substitutions were detected in the MSP119 gene of P. yoelii, a rodent malaria parasite (15). These substitutions are immunologically relevant because protective antibodies generated by vaccination are directed at these polymorphic epitopes (16).

In P. falciparum, a human malaria parasite, five amino acid substitutions have been described in the 19 kDa fragment of MSP1. On the basis of these five possible substitutions, seven natural allelic forms have been described (17–19). It is well established that there are strain-specific as well as conserved epitopes detected by mouse monoclonal antibodies or human polyclonal antibodies from individuals who had been infected with malaria (20–24). It is currently unknown whether protective antibodies induced by vaccination with P. falciparum MSP119 recognize polymorphic or conserved epitopes, or both.

Sequence data for the MSP119 gene from P. vivax (PvMSP119), the second-most important human malaria parasite, are more limited. Deduced amino acid sequences of PvMSP119 from 20 isolates from Southeast Asia and two strains from Latin America showed only a single amino acid substitution (25). However, it is unknown whether this single substitution is of immunological significance.

Recently, we have evaluated several immunoepidemiological aspects of the naturally acquired human immune responses to the MSP1 of P. vivax, the parasite that accounted for 75% of the 405,000 cases of malaria reported in Brazil in 1997. We found that PvMSP119 was the most immunogenic of 11 recombinant proteins during natural infection in humans, being recognized by antibodies or T cells of 83.8% of individuals recently exposed to P. vivax. Even more important was the finding that the antibody titers to PvMSP119 were higher than the titers to other recombinant proteins representing the N-terminal region of PvMSP1 (26,27). This high frequency of responders to PvMSP119 was also described in an independent survey performed in an area of P. vivax transmission in New Guinea (28). The fact that more than 70% of the individuals had IgG antibodies suggests that PvMSP119 is highly conserved among the different isolates of P. vivax. Nevertheless, some individuals fail to develop antibodies to PvMSP119 even in the presence of high antibody titers to other polypeptides of PvMSP1 (26,27).

The lack of reactivity observed in some individuals could be explained at least in part by allelic variation. Given this possibility, the present study was designed to examine the extent of PvMSP119 polymorphism in Brazil and its immunological relevance for recognition by antibodies. Initially, we sequenced the DNA corresponding to PvMSP119 of 88 gene fragments obtained from 28 Brazilian isolates. Subsequently, we evaluated the reactivity of rabbit polyclonal antibodies, a monoclonal antibody, and a panel of 80 human sera with bacterial and yeast-derived recombinant proteins representing the two allelic forms of PvMSP119 described thus far.

Materials and Methods

Polyclonal and Monoclonal Antibodies

The polyclonal rabbit antiserum was obtained by immunization of a rabbit with a baculovirus-derived recombinant protein representing the 19 kDa region of PvMSP1 (29,30) in Freund’s adjuvant (J. Barnwell, unpublished data). The IgG fraction was purified by affinity chromatography using a protein A-agarose matrix (Pharmacia). Antibody concentration was estimated by optic density at 260 nm. The initial concentration was 1 mg/ml. Murine anti-PvMSP119 monoclonal antibody (MAb) 3F8.A2 and mouse ascites were produced as described by Barnwell et al. (31).

Parasites and Human Blood Samples

After receiving each patient’s informed consent, we obtained blood samples from 80 patients with vivax malaria who were diagnosed at Instituto Evandro Chagas in Belém, state of Pará, in the northern part of Brazil. Details on the study area, patient age, and diagnosis have been described by Soares et al. (26,27). Eighteen additional blood samples were collected at the Tropical Medical Center (CEMETRON), Porto Velho, state of Rondônia, in the northwest part of Brazil. Five milliliters of peripheral blood were collected from each patient directly into EDTA tubes before drug treatment. Plasma and infected erythrocytes were removed after centrifugation, stored frozen at −20°C, and transported to São Paulo for further study.

Cloning of PCR Products and Sequencing

Genomic DNA was purified from infected blood samples as described earlier (32). Briefly, 1 ml of TE (10 mM Tris-HCl, pH 7.4, and 1 mM EDTA, pH 8.0) was added to each blood sample. After centrifugation at 12,500 × g for 10 sec, the pellet was resuspended a second time in TE, and the procedure was repeated twice. The colorless pellets were resuspended in 200 µl of PK buffer (20 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl2, and 10 mg/ml proteinase K). Samples were incubated at 65°C for 45 min and then at 95°C for 15 min to inactivate proteinase K. Samples were stored at 4°C until needed. Amplification reactions of the C-terminal region of the PvMSP1 gene (314 bp) were performed using primers 5′-GCTAAATGTGCAAACTCAGTTATTA-3′ (forward) and 5′ — AGCTTAGGAAGCTGGAGGAGCTACA-3′ (reverse) purchased from Life Technologies. For the N-terminal region, we used the primers described by Porto et al. (33), kindly provided by Dr. H. A. del Portillo (ICB, University of São Paulo). Polymerase chain reaction (PCR) mixtures were as follows: 5 µl of blood DNA; 20 mM Tris-HCl, pH 8.4; 50 mM KCl; 1.5 mM MgCl2; 200 µM dATP, dCTP, dGTP, and dTTP; 2.5 U of Taq polymerase (Life Technologies); and 20 pmol of each primer. Amplification reactions were performed with the aid of a Pharmacia thermal cycler (95°C/1 min, 50°C/1 min, 72°C/2 min for 35 cycles). As positive control, PCR reactions were also performed using a cloned PvMSP119 sequence as the PCR template. The PCR products were separated on agarose gel. The 314 bp band was excised from the gel and the DNA purified with glass beads of a commercially available kit (Nucleiclean, Sigma) according to the specifications of the manufacturer. The DNA concentration was estimated in agarose gel. PCR products were cloned directly using a commercially prepared cloning plasmid with a single T-base overhang (pMOS Blue, Amersham). Eighty-eight amplified gene fragments cloned from 28 isolates were selected for sequencing studies. Two to four Plasmids were sequenced from each isolate. Before sequencing, all 88 plasmids were checked for the presence of an insert that hybridized by Southern blot with a radiolabeled DNA representing the PvMSP119 (Belém strain). Nucleotide sequence analysis was done with the thermosequenase cycle sequencing kit (Amersham) according to the manufacturer’s instructions. Plasmid-specific primers (M13 Universal and T7) were labeled with [γ-32P] ATP (Amersham) using T4 polynucleotide kinase (Life Technologies).

Generation of Plasmids Coding for GST-Fusion Proteins Representing Allelic Forms of PvMSP1 19

A recombinant protein containing amino acids 1615 to 1704 of PvMSP119 (Belém strain) was generated. The C-terminal region of PvMSP1 was amplified by PCR, using the cloned PvMSP119 (26) sequence as a substrate for primers: 5′-GGGATCCCCACTATGAGCTCCGAGCAC-3′ (forward) and 5′-TGAATTCCCGCTACAGAAAACTCCCTCAAA-3′ (reverse). The amplified fragment was purified and cloned into the pMOS Blue T-vector, and both strands were sequenced as described above. The insert was removed by treatment with BamHI and EcoRI and ligated into the pGEX-3X expression vector (Pharmacia) treated with these same enzymes. The recombinant GST-PvMSP119 differs from the recombinant protein ICB10 (26) in that it does not contain the amino acids that are most likely to be removed for insertion of the glycerolphosphatidylinositol anchor. This recombinant protein presented a much better solubility and improved yield for purification.

The coding sequence of PvMSP119 (Belém strain) was modified by PCR mutagenesis as follows: a single substitution was introduced, a G for an A (nt 5050), resulting in an amino acid change from Lys1684 to Glu1684 substitution toward the C-terminal of the second epidermal growth factor (EGF) domain. Two PCR amplifications were performed using the following oligonucleotide primers: (i) 5′-GGGATCCCCACTATGAGCTCCGAGCAC-3′ (forward) and 5′-AGACGATTTCATTGCTGTCC-3′ (reverse); (ii) 5′ — GGACAGCAATGAAATCGTCT-3′ (forward) and 5′-TGAATTCCCGCTACAGAAAACTCCCTCAAA-3′ (reverse). Products of these two PCR amplifications were purified on agarose gel and used as targets for a second amplification. The primers for the amplification were 5′-GGGATCCCCACTATGAGCTCCGAGCAC-3′ (forward) and 5′-TGAATTCCCGCTACAGAAAACTCCCTCAAA-3′ (reverse). The product of this PCR reaction (1615Thr to 1704Ser) was purified and cloned into the pMOS Blue T-vector and both strands were sequenced to confirm the single base pair mutation. The insert corresponding to the PCR product was excised by treatment with BamHI and EcoRI and ligated into the pGEX-3X expression vector treated with these same enzymes.

The DNA representing the PvMSP119 fragments of Belém and Asian strains were removed from the pGEX plasmid by treatment with the restriction enzymes Msc1 and Pst1. This DNA fragment was purified and ligated in the presence of dNTPs and Klenow polymerase into the yeast expression plasmid pEG(KT) (34) previously digested with Msc1 and HindIII. Recombinant Plasmids were screened for the presence of the insert by hybridization using [α-32P] ATP-labeled insert.

The correct orientation was determined by restriction analysis.

Expression and Purification of Recombinant Proteins

PvMSP119 representing the Belém and Asian strains were expressed in E. coli (DH5α) as GST fusion proteins essentially as described by Soares et al. (26,27). Expression in the yeast Saccharomyces cerevisiae was performed essentially as described by Mitchell et al. (34). Recombinant proteins and GST were affinity purified on glutathione-Sepharose 4B columns (Pharmacia), their purity was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and protein concentration was estimated by the Bradford method (Biorad).

Immunoassays

Detection of antigen-specific IgG antibodies was done by ELISA. Plasma or serum samples were tested for reactivity with the PvMSP119 recombinant proteins (Belém and Asia) by ELISA as described previously (26,27). Briefly, microtiter plates (Costar, Cambridge, MA) were coated with 200 ng/well of affinity-purified bacterial fusion proteins or GST. Because of restrictions in the amount of yeast-derived recombinant proteins, we used 100 ng/well of these fusion proteins or GST. Human plasma samples were added to wells at 1:200 dilution with subsequent 2-fold serial dilutions until 1:102,400 was attained. Rabbit and mouse serum samples were also tested at the indicated dilutions. After 2 hr incubation at room temperature, unbound material was washed away, and peroxidase-conjugated goat anti-human IgG (Fc-specific, 1:10,000) (Sigma), or anti-rabbit IgG or anti-mouse IgG (both diluted 1:4000, from Kirkegaard and Perry, Gaithersburg, MD) was added to each well. After 1 hr incubation at room temperature, excess labeled antibody was removed during washing, and the reaction was developed with o-phenyl-enediamine (Sigma). Plates were read at 492 nm on an ELISA reader (Labsystems Multiskan MS). The OD492 values in Figure 4 represent binding of IgG to the recombinant protein after subtraction of binding of the same serum to GST alone.

The inhibition assay was performed as described by Devey et al. (35). ELISA plates were coated with the recombinant bacterial PvMSP119 representing the Belém allele. In Figure 5a, the indicated recombinant fusion protein or GST was added at different concentrations (2-fold dilutions ranging from 10 µg/ml to 3 ng/ml) in each well. Human serum at the indicated final dilution was added after the soluble protein. The ELISA was then performed as described above. In Figure 5B, serum samples were diluted to provide 0.75 to 1.25 OD492. The soluble recombinant proteins of PvMSP119 or GST were added to reach a final concentration of 10 µg/ml. The percentage of inhibition for each serum sample was calculated as follows:

$$1 - {{{\rm{O}}{{\rm{D}}_{492}}\;{\rm{in}}\;{\rm{the}}\;{\rm{presence}}\;{\rm{of}}\;{\rm{soluble}}\;{\rm{recombinant}}\;{\rm{protien}}} \over {{\rm{O}}{{\rm{D}}_{492}}\;{\rm{in}}\;{\rm{the}}\;{\rm{absence}}\;{\rm{of}}\;{\rm{soluble}}\;{\rm{recombinant}}\;{\rm{protien}}}} \times 100.$$

Results

Analysis of Polymorphism of PvMSP119 Sequences in 28 Isolates of P. vivax Collected in Different Endemic Areas of Brazil

To estimate the polymorphism of PvMSP119 in Brazil, total DNA was extracted from blood samples of 28 individuals during patent P. vivax infection. Seven of these 28 individuals were permanent residents of the city of Belém, Pará, where they acquired the disease (26,27). Three individuals were infected in the state of Maranhão, and they were diagnosed with malaria in the city of Belém. The states of Pará and Maranhão are located contiguously in the northern part of the Amazon region. Eighteen blood samples were collected from residents of Porto Velho in the state of Rondônia. This area is located in the northwest section of Brazil, more than 1000 miles from the other endemic areas (36).

A 314 bp fragment representing the C-terminus of the P. vivax MSP1 gene was amplified by PCR as described in Materials and Methods. Two to four clones derived from each isolate were sequenced. A total of 88 amplified cloned gene fragments were compared with the original Belém sequence of the P. vivax MSP1 gene. We found that the sequences encoding the PvMSP119 in Brazil were conserved. The dimorphism of PvMSP119 reported in isolates from Asia (25) was not detected in the 28 isolates obtained in Brazil. Also, we were unable to find any other nucleotide substitution.

In parallel, as a control, we amplified the DNA portion coding for the highly polymorphic region P5 located in the N-terminal portion of PvMSP1 (33). According to the size of the PCR products, we found two distinct alleles described earlier in Brazilian isolates (33). Also relevant is the fact that in approximately 40% of the blood samples, we observed the presence of both alleles, indicating that these individuals had mixed infection with at least two strains of P. vivax (data not shown).

Recognition by Antibodies of Two Allelic Forms of PvMSP119 Expressed as Recombinant Bacterial or Yeast GST Fusion Proteins

To determine whether the allelic polymorphism described for PvMSP119 could modify recognition by specific antibodies, a gene encoding PvMSP119 was generated by introducing a single base pair substitution, a G for an A, resulting in an amino acid change from Lys1684 to Glu1684 toward the C-terminus of the second EGF-like domain (Fig. 1A). This gene represents the Asian dimorphic sequence of PvMSP119 (25) and was designated PvMSP119 Asia.

Fig. 1
figure 1

Plasmid DNA sequence analysis and SDS-PAGE of recombinant proteins representing the two allelic forms of PvMSP119. (A) The single base pair difference between the two allelic forms of PvMSP119 is indicated in a sequencing gel. (B, C) SDS-PAGE of purified recombinant fusion proteins or GST produced in E. coli and S. cerevisiae, respectively. Lane 1, GST protein; lane 2, GST fusion protein representing PvMSP119 Belém; lane 3, GST fusion protein representing PvMSP119 Asia. Molecular weight standard (MWSTD) values are expressed in kDa.

Full size image

PvMSP119 recombinant proteins representing the Belém and Asia strains were expressed as soluble GST fusion proteins of ~38 kDa in E. coli and S. cerevisiae. As determined by SDS-PAGE, the recombinant GST-MSP119 representing the Asian allele migrates with a molecular weight that is slightly higher than that of the GST fusion protein representing the MSP119 allele from Belém strain (Fig. 1B, C).

Initially, these four fusion proteins and GST were used as antigens for recognition by a murine MAb specific for PvMSP119 (31) or by IgG purified from a rabbit immunized with a baculo-virus-expressed recombinant PvMSP119 [Belém strain (29,30)]. Both the polyclonal antiserum and the MAb strongly inhibited the in vitro invasion of reticulocytes by P. vivax merozoites of the Belém strain (J. Barnwell, unpublished results). We found that rabbit polyclonal antibodies recognized similarly the recombinant proteins representing each allelic form of PvMSP119 generated in E. coli (Fig. 2A). Comparable recognition was also observed when we used yeast-derived recombinant proteins in the ELISA (Fig. 2B). The MAb was also unable to differentiate the two allelic forms of PvMSP119 produced either in E. coli or in yeast (Fig. 2C, D, respectively).

Fig. 2
figure 2

Recognition of recombinant proteins representing both allelic forms of PvMSP119 by rabbit polyclonal antibodies and a mouse MAb. (A, B) Antibody titration curves using rabbit polyclonal antibodies specific for PvMSP119. ELISA plates were coated with recombinant proteins produced in bacteria (A) or yeast (B). (C, D) Antibody titration curves using mouse MAb specific for PvMSP119. ELISA plates were coated with recombinant proteins produced in bacteria (C) or yeast (D). Results are shown as the average of triplicate samples ± standard deviation (SD).

Full size image

Next, we evaluated the serological recognition of the recombinant proteins representing the two allelic forms of PvMSP119 by antibodies collected from humans who were infected with P. vivax. In our initial analysis we selected two individuals from Belém. Serum samples were collected from these two individuals during their first episode of P. vivax malaria. These individuals had never been infected with P. vivax or P. falciparum before. From a single blood sample, we obtained serum as well as DNA. The DNA was used as a target for amplification and four cloned PCR products from each individual were sequenced. In both cases, the DNA sequence matched the Belém allele of PvMSP119. By ELISA, we observed that sera collected from each of these two individuals reacted equally well with recombinant proteins representing both allelic forms of PvMSP119 (Fig. 3). The same results were obtained when we used bacterial (Fig. 3A, B) or yeast-derived (Fig. 3C, D) recombinant proteins.

Fig. 3
figure 3

Recognition of recombinant proteins representing both allelic forms of PvMSP119 by antibodies from two individuals during their first infection with P. vivax. Antibody titration curves were calculated for serum samples from two individuals during their first episode of malaria (A, C: individual 40; B, D: individual 51). ELISA plates were coated with recombinant proteins produced in bacteria (A, B) or yeast (C, D). Results are shown as the average of duplicate samples.

Full size image

We then extended this same type of analysis to 78 other serum samples collected from individuals with patent P. vivax infections. In the serum of 77 of the 80 individuals (96.25%), no difference was detected in ELISA titers to each recombinant protein representing the two allelic forms of PvMSP119. When the recognition of the two proteins was directly compared at different serum dilutions, there was a high degree of correlation between them (r2 > 0.97 in all cases; Fig. 4). Only 3 of the 80 serum samples (3.75%) displayed slightly increased reactivity with a recombinant protein representing one of the allelic forms. One of them recognized the Belém allele better than the Asian allele and the other two displayed a slightly higher reactivity with protein representing the Asian allele. Although we reproduced this analysis on different occasions, the difference in antibody titers represented only a single, 2-fold dilution (data not shown).

Fig. 4
figure 4

Comparison of antibody recognition of bacterial recombinant proteins representing both allelic of PvMSP119. Each panel represents the reactivity at different dilutions of serum samples obtained from 80 individuals who were infected with P. vivax. Results are shown as the average of duplicate samples The correlation coefficient (r2) for each graph is shown at the bottom.

Full size image

Similar analysis was also performed with yeast-derived recombinant proteins in the ELISA. We found a very high correlation between both proteins representing the two allelic forms of PvMSP119 in their serum recognition (r2 = 0.96, n = 30, data not shown). ELISAs were also performed using subclass-specific antibodies to human IgG1 and IgG3, the predominant subclasses of IgG to PvMSP119 (26,27). The correlation observed was also high (r2 = 0.936 and 0.875 for IgGl and IgG3, respectively, n = 34; data not shown).

Finally, we performed inhibition assays with bacterial recombinant proteins representing both allelic forms of PvMSP119. An example of the inhibitory curve obtained with each recombinant protein is shown in Figure 5A. Different concentrations of both proteins inhibited human antibody binding to PvMSP119 Belém at exactly the same extension. Inhibitory assays were performed with 50 serum samples from P. vivax infected individuals. In most individuals (64%), inhibition of binding was very high (>90%) for soluble recombinant proteins representing each allele of PvMSP119. These individuals are indicated in Figure 5B as group I. In a smaller group of individuals (36%), we observed the presence of allele-specific antibodies. In these cases, the recombinant protein PvMSP119 Asia was unable to inhibit the binding of human antibodies to the same extension as the protein PvMSP119 Belém (group II). Nevertheless, in these individuals the average inhibition obtained by proteins PvMSP119 Belém and PvMSP119 Asia were 94.30 ± 6.0 and 77.09 ± 7.7%, respectively, indicating that most of the antibodies were directed to conserved epitopes present in both allelic forms. To confirm the presence of allele-specific antibodies, we inhibited the binding of sera from individuals of group II to wells coated with PvMSP119 Asia. We observed that both recombinant proteins inhibited the binding at a similar extension. The values of inhibition to GST, PvMSP119 Asia, and PvMSP119 Belém were 2.3 ± 3.8, 91.4 ± 7.8, and 89.5 ± 9.7%, respectively. These findings confirm that PvMSP119 Belém displays allele-specific epitopes.

Fig. 5
figure 5

Inhibition ELISA carried out with recombinant proteins representing each allelic form of PvMSP119. ELISA plates were coated with a bacterial recombinant protein representing PvMSP119 Belém allele. (A) The indicated recombinant fusion protein or GST was added at different concentrations in each well. Human serum from individual 40 was added at a dilution of 1:800. Results are expressed as the average of triplicate samples ± SD. (B) Average inhibition of the binding of human sera to recombinant protein PvMSP119 Belém. The indicated soluble recombinant proteins or GST was added to a final concentration of 10 µg/ml. The percentage of inhibition was calculated as described in Materials and Methods, individuals were selected for group I when inhibition values with either recombinant protein were >90%. Group II represents individuals who had inhibition of <90% when the recombinant protein PvMSP119 Asia was used. Results are expressed as the average of the indicated number of individuals ± SD.

Full size image

In similar experiments using the rabbit polyclonal antibodies to PvMSP119, we observed that both proteins inhibited more than 96% of the antibody binding to PvMSP119 Belém or to PvMSP119 Asia (data not shown).

Discussion

Our study on the variability of the DNA sequences encoding MSP119 in Brazilian isolates of P. vivax failed to identify allelic forms distinct from the originally described Belém allele. Our data contrasted with the results from studies performed in Southeast Asia where two allelic forms of PvMSP119 were found (25). It is possible that this variant also exists in Brazil. However, the Belém allele predominated in isolates collected in the two major endemic areas of the country. In Asia, the variant allelic form was very frequent, representing 9 of the 20 isolates sequenced.

The conservation found thus far in the sequences of PvMSP119 is unexpected. It seems to be restricted to the C-terminal region because a much wider sequence variation has been described for the N-terminal region of PvMSP1 (37–39). Also, this conservation is restricted to P. vivax, since distinct allelic forms of MSP119 have been found in Brazilian isolates of P. falciparum (M.U. Ferreira, unpublished results). The PvMSP119 sequence conservation contrasts sharply with data of MSP119 sequences obtained from other species of malaria parasites such as P. yoelii and P. falciparum. In both cases, several allelic forms have been found in natural isolates. In view of the variation found in other species, the conservation in PvMSP119 may suggest a strong selective pressure to maintain the structure and imply a function for this part of the molecule.

The reason for the allelic dimorphism described for PvMSP119 in Asia is presently unknown. A reasonable explanation for such variability is that PvMSP119 provides a mechanism for the parasite to evade the antibody response. If this were the case, the polymorphism would raise doubts about the possible use of PvMSP119 as part of subunit vaccines against P. vivax. We assessed this question by studying the recognition by antibodies of recombinant proteins representing the two allelic forms of the protein. We found that recombinant proteins representing both allelic forms produced in two distinct expression systems (bacterial and yeast) were equally well recognized by specific antibodies that inhibit merozoite invasion in vitro. Also, a strikingly similar recognition was observed with a panel of 80 human sera from infected individuals who were exposed to P. vivax. These results suggest that the single amino acid modification found thus far in nature does not impair the recognition of PvMSP119 by IgG antibodies of most naturally infected individuals.

Because we used recombinant proteins, it is not possible to completely rule out that allele specific epitopes are expressed more in the native forms of PvMSP119. This possibility can be correctly addressed in the future by active immunization of experimental animals with each one of the allelic forms followed by a challenge with blood stages of P. vivax (Belém or Asia strains).

Earlier studies on the recognition of different allelic forms of MSP119 were performed in two other species of Plasmodium. The results obtained with the rodent malaria agent P. yoelii unequivocally demonstrated that protective antibodies are directed mainly at the polymorphic residues of MSP119. Antibodies from mice immunized with a bacterial recombinant protein representing a certain allele of MSP119 only recognized parasites of a homologous strain. Protective immunity against P. yoelii was also strain-specific (16). This work suggested that similar strain-specific protective antibodies could be generated in human malaria.

Studies on the recognition of allelic forms of MSP119 of P. falciparum, a human malaria parasite, were performed with yeast-derived recombinant proteins representing four alleles (E-KNG, Q-KNG, E-TSR, and Q-TSR). They demonstrated that there are multiple conserved epitopes among these different allelic forms recognized by human and non-human primate antibodies (23,24,40–42). The recognition by human antibodies of recombinant proteins representing allelic forms with a single amino acid modification (E-KNG and Q-KNG) was quite similar (23,40). These results resemble the findings reported here, and they may suggest that single amino acid substitutions may not be sufficient to impair recognition by antibodies. The recognition of a third allelic form (E-TSR), which has three or four amino acid modifications compared to E-KNG or Q-KNG, respectively, was not as similar, and the presence of strain-specific human antibodies was observed (23,24,40). To our knowledge, no comparisons between other allelic forms found in nature have been reported thus far (19).

Alternatively, the single amino acid substitution found in the two allelic forms of PvMSP119 may represent a strategy to evade cell-mediated immune responses. It is well documented that few amino acid changes can dramatically impair the binding of peptides to MHC class I or class II (43–46). They may also reduce recognition by T cells or generate antagonistic peptides that inhibit activation of specific T cells by the MHC-peptide complex (47–49). It is possible that in Asia there may be individuals with a particular MHC that recognize exclusively one of the two allelic forms. In this case, the dimorphism could be an advantage to the parasite. Although we have described the presence of T cell epitopes in PvMSP119 (26), this possibility will have to be further explored in transmission areas where both allelic forms of PvMSP119 are found.

Finally, the dimorphism may represent an adaptation to host polymorphism, for example, allowing the MSP1 molecule to bind to a polymorphic reticulocyte receptor. This possibility cannot be immediately addressed because an MSP1 receptor has not been described.

In conclusion, the present findings suggest that the use of PvMSP119 as part of a subunit vaccine against P. vivax might be greatly facilitated by the limited genetic polymorphism and the predominant recognition of conserved epitopes by antibodies.

References

  1. Good MF, Kaslow DC, Miller LH. (1998) Pathways and strategies for developing a malaria blood-stage vaccine. Annu. Rev. Immunol. 16: 57–87.

    Article  CAS  Google Scholar 

  2. Blackman MJ, Heidrich HG, Donachie S, McBride JS, Holder AA. (1990). A single fragment of a malaria merozoite surface protein remains on the parasite during red blood cell invasion and is the target of invasion-inhibiting antibodies. J. Exp. Med. 172: 379–382.

    Article  CAS  Google Scholar 

  3. Pirson PJ, Perkins ME. (1985) Characterization with monoclonal antibodies of a surface antigen of Plasmodium falciparum merozoites. J. Immunol. 134: 1946–1951.

    CAS  PubMed  Google Scholar 

  4. Chappel JA, Holder AA. (1993) Monoclonal antibodies that inhibit Plasmodium falciparum invasion in vitro recognise the first growth factor-like domain of merozoite surface protein-1. Mol. Biochem. Parasitol. 60: 303–312.

    Article  CAS  Google Scholar 

  5. Chang SP, Gibson HL, Lee NC, Barr PJ, Hui GS. (1992) A carboxyl-terminal fragment of Plasmodium falciparum gp 195 expressed by a recombinant baculovirus induces antibodies that completely inhibit parasite growth. J. Immunol. 149: 548–555.

    CAS  PubMed  Google Scholar 

  6. Burns JM, Parke LA, Daly TM, Cavacini LA, Weidanz WP, Long CA. (1989) A protective monoclonal antibody recognizes a variant-specific epitope in the precursor of the major merozoite surface antigen of the rodent malarial parasite Plasmodium yoelii. J. Immunol 142: 2835–2840.

    CAS  PubMed  Google Scholar 

  7. Daly TM, Long CA. (1995) The humoral immune response to a carboxyl-terminal region of the merozoite surface protein-1 plays a predominant role in controlling blood-stage infection in rodent malaria. J. Immunol 155: 236–243.

    CAS  PubMed  Google Scholar 

  8. Daly TM, Long CA. (1993) A recombinant 15-kilodalton carboxyl-terminal fragment of Plasmodium yoelii 17XL merozoite surface protein-1 induces a protective immune response in mice. Infect. Immun. 61: 2462–2467.

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Ling IT, Ogun SA, Holder AA. (1994) Immunisation against malaria with a recombinant protein. Parasite Immunol. 16: 63–67.

    Article  CAS  Google Scholar 

  10. Kumar S, Yadava A, Keister DB, et al. (1995) Immunogenicity and in vivo efficacy of recombinant Plasmodium falciparum merozoite surface protein-1 in Aotus monkeys. Mol Med. 1: 325–332.

    Article  CAS  Google Scholar 

  11. Tian JH, Kumar S, Kaslow DC, Miller LH. (1997) Comparison of protection induced by immunization with recombinant proteins from different regions of merozoite surface protein 1 of Plasmodium yoelii. Infect. Immun. 65: 3032–3036.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Perera KLRL, Handunnetti SM, Holm I, Longacre S, Mendis K. (1998) Baculovirus merozoite surface protein 1 C-terminal recombinant antigens are highly protective in a natural primate model for human Plasmodium vivax malaria. Infect. Immun. 66: 1500–1506.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Yang C, Collins WE, Sullivan JS, Kaslow DC, Xiao L, Lal AA. (1999) Partial protection against Plasmodium vivax blood-stage infection in Saimiri monkeys by immunization with a recombinant C-terminal fragment of merozoite surface protein 1 in block copolymer adjuvant. Infect. Immun. 67: 342–349.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Ling IT, Ogun SA, Holder AA. (1995) The combined epidermal growth factor-like modules of Plasmodium yoelii merozoite surface protein-1 are required for a protective immune response to the parasite. Parasite Immunol 17: 425–433.

    Article  CAS  Google Scholar 

  15. Daly TM, Burns JM, Long CA. (1992) Comparison of the carboxy-terminal, cysteine-rich domain of the merozoite surface protein-1 from several strains of Plasmodium yoelii. Mol. Biochem. Parasitol 52: 279–282.

    Article  CAS  Google Scholar 

  16. Rénia L, Ling IT, Marussig M, Miltgen F, Holder AA, Mazier D. (1997) Immunization with a recombinant C-terminal fragment of Plasmodium yoelii merozoite surface protein 1 protects mice against homologous but not heterologous P. yoelii sporozoite challenge. Infect. Immun. 65: 4419–4423.

    PubMed  PubMed Central  Google Scholar 

  17. Tanabe K, MacKay M, Goman M, Scaife JG. (1987) Allelic dimorphism of a surface antigen gene of the malaria parasite. J. Mol. Biol. 134: 1946–1951.

    Google Scholar 

  18. Miller LH, Roberts T, Shahabuddin M, McCutchan T. (1993) Analysis of the sequence diversity in the Plasmodium falciparum merozoite surface protein-1. Mol Biochem. Parasitol. 59: 1–14.

    Article  CAS  Google Scholar 

  19. Qari SH, Shi YP, Goldman IF, Nahlen BL, Tibayrenc M, Lal AA. (1998) Predicted and observed alleles of Plasmodium falciparum merozoite surface protein-1 (MSP-1), a potential malaria vaccine antigen. Mol. Biochem. Parasitol. 92: 241–252.

    Article  CAS  Google Scholar 

  20. Wilson CF, Anand R, Clark JT, McBride JS. (1987) Topography of epitopes on a polymorphic antigen of Plasmodium falciparum determined by the binding of monoclonal antibodies in a two-site radioimmunoassay. Parasite Immunol 9: 737–746.

    Article  CAS  Google Scholar 

  21. McBride JS, Heidrich HG. (1987) Fragments of the polymorphic Mr 185,000 glycoprotein from the surface of isolated Plasmodium falciparum merozoites form antigenic complex. Mol. Biochem. Parasitol. 23: 71–84.

    Article  CAS  Google Scholar 

  22. Cooper JA, Cooper LT, Saul AJ. (1992) Mapping of the region predominantly recognized by antibodies to Plasmodium falciparum surface antigen MSA 1. Mol. Biochem. Parasitol. 51: 301–312.

    Article  CAS  Google Scholar 

  23. Udhayakumar V, Anyona D, Kariuki S, et al. (1995) Identification of T and B cell epitopes recognized by humans in the C-terminal 42-kDa domain of Plasmodium falciparum merozoite surface protein (MSP)-l. J. Immunol. 154: 6022–6030.

    CAS  PubMed  Google Scholar 

  24. Shi YP, Sayed U, Qari SH, et al. (1996) Natural immune response to the C-terminal 19-kilodalton domain of Plasmodium falciparum merozoite surface protein 1. Infect. Immun. 64: 2716–2723.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Pasay MC, Cheng Q, Rzepczyk C, Saul A. (1995) Dimorphism of the C terminus of the Plasmodium vivax merozoite surface protein 1. Mol Biochem. Parasitol. 70: 217–219.

    Article  CAS  Google Scholar 

  26. Soares IS, Levitus G, Souza JM, del Portillo HA, Rodrigues MM. (1997) Acquired immune responses to the N- and C-terminal regions of Plasmodium vivax merozoite surface protein 1 in individuals exposed to malaria. Infect. Immun. 65: 1606–1614.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Soares IS, Cunha MG, Silva MN, Souza JM, del Portillo HA, Rodrigues MM. (1999) Longevity of the naturally acquired antibody responses to the N- and C-terminal regions of Plasmodium vivax MSP1. Am. J. Trop. Med. Hyg. 60: 357–363.

    Article  CAS  Google Scholar 

  28. Fraser T, Michon P, Barnwell JW, et al. (1997) Expression and serologic activity of a soluble recombinant Plasmodium vivax Duffy binding protein. Infect. Immun. 65: 2772–2777.

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Longacre S, Mendis KN, David P. (1994) Plasmodium vivax merozoite surface protein 1 recombinant proteins in baculovirus. Mol Biochem. Parasitol. 64: 191–205.

    Article  CAS  Google Scholar 

  30. Holm I, Nato F, Mendis KN, Longacre S. (1997) Characterization of the C-terminal merozoite surface protein 1 baculovirus recombinant proteins from Plasmodium vivax and Plasmodium cynomolgi as recognized by natural anti-parasite immune response. Mol. Biochem. Parasitol. 89: 313–319.

    Article  CAS  Google Scholar 

  31. Barnwell JW, Galinski MR, DeSimone SG, Perler F, Ingravallo P. (1999) Plasmodium vivax, P. cynomolgi, P. knowlesi: identification of homologue proteins associated with the surface of merozoites. Exp. Parasitol. 91: 238–249.

    Article  CAS  Google Scholar 

  32. del Portillo HA, Parrillo LB, Camargo AA, Levitt A. (1993) Amplification of single-copy genes of Plasmodium vivax from two drops of peripheral blood of infected patients. J. Protozool. Res. 3: 20–24.

    Google Scholar 

  33. Porto M, Ferreira MU, Camargo LMA, Premawansa S, del Portillo HA. (1992) Second form in a segment of the merozoite surface protein 1 gene of Plasmodium vivax among isolates from Rondônia (Brazil). Mol. Biochem. Parasitol. 54:121–124.

    Article  CAS  Google Scholar 

  34. Mitchell DA, Marshall TK, Deschenes RI. (1993) Vectors for the inducible overexpression of glutathione S-transferase fusion proteins in yeast. Yeast 9: 715–723.

    Article  CAS  Google Scholar 

  35. Devey ME, Bleasdale K, Lee S, Rath S. (1988) Determination of the functional affinity of IgG1 and IgG4 antibodies to tetanus toxoid by isotype-specific solid-phase assays. J. Immunol. Methods 106: 119–125.

    Article  CAS  Google Scholar 

  36. Camargo LMA, Dal Colletto GMD, Ferreira MU, et al. (1996) Hypoendemic malaria in Rondonia (Brazil, western Amazon region): seasonal variation and risk groups in an urban locality. Am. J. Trop. Med. Hyg. 55: 32–38.

    Article  CAS  Google Scholar 

  37. del Portillo HA, Longacre S, Khouri E, David P. (1991) Primary structure of the merozoite surface antigen 1 of Plasmodium vivax reveals sequences conserved between different Plasmodium species. Proc. Natl. Acad. Sci. U.SA. 88: 4030–4034.

    Article  Google Scholar 

  38. Gibson HL, Tucker JE, Kaslow DC, et al. (1992) Structure and expression of the gene for Pv200, a major blood-stage surface antigen of Plasmodium vivax. Mol. Biochem. Parasitol. 50: 325–334.

    Article  CAS  Google Scholar 

  39. Kirchgatter K, del Portillo HA. (1998) Molecular analysis of Plasmodium vivax relapses using the MSP1 molecule as a genetic marker. J. Infect. Dis. 177: 511–515.

    Article  CAS  Google Scholar 

  40. Egan AF, Chappel JA, Burghaus PA, et al. (1995) Serum antibodies from malaria-exposed people recognize conserved epitopes formed by the two epidermal growth factor motifs of MSP119, the carboxy-terminal fragment of the major merozoite surface protein of Plasmodium falciparum. Infect. Immun. 63: 456–466.

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Kaslow DC, Hui G, Kumar S. (1994) Expression and antigenicity of Plasmodium falciparum merozoite surface protein 1 (MSP19) variants in Saccharomyces cerevisiae. Mol. Biochem. Parasitol. 63: 283–289.

    Article  CAS  Google Scholar 

  42. Hui GSN, Nikaido C, Hashiro C, Kaslow DC, Collins WE. (1996) Dominance of conserved B-cell epitopes of the Plasmodium falciparum merozoite surface protein, MSP1, in blood-stage infections of naive Aotus monkeys. Infect. Immun. 64: 1502–1509.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Koup R. (1994) Virus escape from CTL. J. Exp. Med. 180: 779–782.

    Article  CAS  Google Scholar 

  44. Couillin I, Culman-Penciolelli B, Gomard E, Levy J-P, Guillet J-G, Saragosti S. (1994) Impaired CTL recognition due to genetic variations in the main immunogenic region of HTV-1 nef protein. J. Exp. Med. 180: 1129–1134.

    Article  CAS  Google Scholar 

  45. de Campos Lima P-O, Levistky V, Brooks J, et al. (1994) T cell response and virus evolution: loss of HLA-A11-restricted CTL epitopes in Epstein-Barr virus isolates from highly All-positive populations by selective mutations at the anchor sequence. J. Exp. Med. 179: 1297–1305.

    Article  Google Scholar 

  46. Udhayakumar V, Ongecha JM, Shi YP, et al. (1997) Cytotoxic T cell reactivity and HLA-B35 binding of the variant Plasmodium falciparum circunsporozoite protein CD8+ CTL epitope in naturally exposed Kenian adults. Eur. J. Immunol. 27: 1952–1957.

    Article  CAS  Google Scholar 

  47. Pircher H, Moskophidis D, Roher U, Burki K, Hengartner H, Zinkernagel DM. (1990) Viral escape by selection of cytotoxic T-cell resistant virus variants in vivo. Nature 34: 629–633.

    Article  Google Scholar 

  48. Bertoletti A, Sette A, Chisari FV, et al. (1993) Natural variants of cytotoxic epitopes are T-cell receptor antagonists for antiviral cytotoxic T cells. Nature 364: 158–160.

    Article  Google Scholar 

  49. Gilbert SC, Plebanski M, Gupta S, et al. (1998) Association of malaria parasite, population structure, HLA, and immunological antagonism. Science 279: 1173–1177.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors are indebted to Dr. Hernando Del Portillo, University of São Paulo, for providing the plasmid ICB10. This work was supported by grants from FAPESP, PADCT, CNPq, PRONEX, and FINEP to M.M.R. M.U.F. and M.M.R. are the recipients of fellowships from CNPq. I.S.S. is a post-doctoral fellow of FAPESP.

Author information

Authors and Affiliations

  1. Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil

    Irene S. Soares, Maristela Gomes Da Cunha, Beatriz A. Castilho &  Mauricio M. Rodrigues

  2. Departamento de Patologia, Centro de Ciências Biológicas, Universidade Federal do Pará, Belém, Pará, Brazil

    Irene S. Soares & Maristela Gomes Da Cunha

  3. Division of Parasitic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA

    John W. Barnwell

  4. Departamento de Parasitologia, Universidade de São Paulo, São Paulo, Brazil

    Marcelo U. Ferreira & Jomar P. Laurino

Authors
  1. Irene S. Soares
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. John W. Barnwell
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Marcelo U. Ferreira
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Maristela Gomes Da Cunha
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jomar P. Laurino
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Beatriz A. Castilho
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Mauricio M. Rodrigues
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mauricio M. Rodrigues.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Soares, I.S., Barnwell, J.W., Ferreira, M.U. et al. A Plasmodium vivax Vaccine Candidate Displays Limited Allele Polymorphism, Which Does Not Restrict Recognition by Antibodies. Mol Med 5, 459–470 (1999). https://doi.org/10.1007/BF03403539

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF03403539

Molecular Medicine

ISSN: 1528-3658