Background
Malaria is one of the major causes of death from infection in developing countries. Development of an effective malaria vaccine may reduce malaria-associated severe morbidity and mortality in malaria-endemic areas. A number of parasite surface antigens of asexual blood stages are being investigated as vaccine candidate antigens. [
1,
2]. Among these antigens, merozoite surface protein-1 (MSP-1) is a leading candidate antigen [
3]. The
msp-gene encodes a 195 kDa protein that is cleaved in four distinct fragments (83, 28–30, 38–45 and 42 kDa) at the time of schizont rupture. During merozoite invasion, the carboxy-terminal 42-kDa fragment is further processed to yield a 19-kDa fragment (MSP-1
19) which remains associated with merozoites [
4,
5]. A number of vaccination studies with MSP-1
19 and MSP-1
42, in mice and monkeys have shown partial and full protection from malaria infection [
6‐
11]. A substantial proportion of antibodies directed to MSP-1
19 in
Plasmodium falciparum-infected human sera have been shown to inhibit erythrocyte invasion
in vitro [
12]. Importantly, MSP-1
19-mediated protective immune responses are largely antibody dependent with high antibody titres being essential for the protection [
13,
14].
The
msp-1 of
P. falciparum has been shown to be dimorphic, K1/Wellcome and MAD 20 types [
15,
16]. Sequence comparison of
P. falciparum msp-1 sequences among different geographical isolates shows a great deal of variations. Based on sequence analysis,
msp-1 has been divided into 17 blocks comprising of conserved, semi conserved and variable regions [
5,
15]. Intragenic recombination between the two allelic types appears to be the main cause for variability among different field isolates [
16‐
18]. The C-terminal 19 kDa region of
msp-1, that represents the 17
th block, consists of two EGF like domains [
5] and has been shown to be highly conserved among different isolates, with single amino acid substitution at five different positions. These changes are E μ Q at position 1644 in the first EGF domain and at positions, 1691 (T μ K), 1700 (S μ N), 1701 (R μ G) and 1716 (L μ F) [
19]. Based on these variations, several variant forms of PfMSP-1
19 have been described among different
P. falciparum isolates around the world. However, there are a limited number of reports of genetic diversity of C-terminal region of MSP-1
19 in isolates from the Indian subcontinent [
18,
20,
21], that contribute around two million cases every year (Source: National Vector Borne Disease Control Programme).
The present study investigated sequence variations in MSP-119 region among different field isolates from a malaria endemic area in India. This study reveals existence of seven variant types in a single geographical location. In addition, three of the MSP-119 variants, Q-KNG-L, E-KNG-L and E-TSR-F were expressed and the relative abundance of specific antibodies in their respective sera studied.
Methods
Collection of P. falciparum infected blood and sera
P. falciparum infected blood samples were collected on filter paper by finger prick from malaria patients participating in cross-sectional and longitudinal malaria epidemiology surveys being conducted in malaria endemic villages in Sundergarh district, Orissa in eastern India [
23]. Thirteen study villages were chosen, out of which eight villages were located in deep forests and five villages were located in plain areas. Prior consent of the patients and the consent of institutional ethical committee were taken before the beginning of the study. Age of the patients ranged between six months to 17 years. In case of infants, the consent of parents were obtained. All the samples were positive for
P. falciparum infection as determined by Giemsa-stained thick smears examined microscopically. Sixteen field spots were chosen from six different villages and DNA was isolated from these spots by the boiling method [
24]. Blood samples were also collected and allowed to clot at room temperature, and serum was collected by centrifugation at 2,000 rpm for 10 min at 4°C and stored at -20°C until used.
PCR amplification, cloning and sequencing
To determine the sequence polymorphism in PfMSP-119 gene, PCR amplification was done in a 50 μl reaction volume using 10 ng genomic DNA from different P. falciparum isolates and primers MSP1-19F1 (Forward: 5' ATT GAG ACC TTA TAC AAT AAC 3') and MSP1-19R1 (Reverse: 5' TTA AGG TAA CAT ATT TTA ACT CCT AC 3'). The thermocycler profile was 5 min hot start at 94°C and 30 cycles each of 30 sec at 94°C, 1 min at 50°C and 30 sec at 72°C. In some cases, nested PCR was employed using a second set of forward primer MSP1-19F2 (Forward: 5' GAT ACG AAA AAA GAT ATG CTT GG 3') and the same reverse primer. The amplified PCR products (480 bps) were analysed by agarose gel electrophoresis and purified by QIAquick Gel Extraction Kit (Qiagen). The purified PCR fragments were cloned into pGEM-T cloning vector as per manufacturer's instructions (Promega). The positive clones were selected by restriction enzyme analysis. Three independent clones from a minimum of two PCR amplifications for each isolate were sequenced using T7 forward and M13 reverse primers.
Expression and purification of three PfMSP-119 variants
For the expression of PfMSP-119 variants, the 288-bp fragment corresponding to PfMSP-119 region were amplified using primers; 5'-GACTAGGGATCCATTTCACAACACCAATGCG-3' and 5'-GCTGATGTCGACTTAGTTAGAGGAACTGCAGAA-3' and cloned into pQE-30 expression vector in Bam HI and Sal I sites. This vector provides six His residues at the N terminus of the expressed protein. Expression of the recombinant MSP-119 was induced with 0.5 mM IPTG in the Escherichia coli strain M15. For the purification of MSP-119 proteins, the induced pellets were sonicated in 1 × TBS (Tris 50 mM, NaCl 500 mM), pH 8.0 containing 1 mM PMSF and 0.05% Tween 20. The supernatant containing the expressed proteins were incubated with Nickel-NTA-Agarose for 1 h at room temperature. The resins were washed with 1 × TBS containing 10 mM imidazole. The bound proteins were eluted with an imidazole gradient of 20 to 250 mM at pH 8.0. Q-KNG-L variant was eluted with a linear gradient of 10 to 70 mM Imidazole in 20 mM Tris-500 mM NaCl (pH 8.0) buffer, E-KNG-L variant was eluted in a linear gradient of 50–250 mM and E-TSR-F was eluted in 250 mM Imidazole. The eluates were analysed by SDS-PAGE, the fractions containing a clear single protein band were pooled, and the protein concentrations were determined.
ELISA
Reactivity of the recombinant PfMSP-1
19 proteins with patient sera was evaluated by ELISA as described earlier [
25]. Briefly, 96-well microplates (Dynatech) were coated with 50 ng of any of the three recombinant PfMSP-1
19 proteins per well in 0.06 M carbonate-bicarbonate buffer (pH 9.6). The plates were incubated overnight at 4°C, and the wells were blocked with 5% low-fat milk in PBS (pH 7.2) for 1 h at room temperature. The antigen-coated wells were sequentially incubated with serial dilutions (starting from 1:50 dilutions) of the individual patient sera and optimally diluted enzyme-labelled secondary antibody (horseradish peroxidase-labeled antihuman immunoglobulin, IgG). In between these incubations, the plates were washed with a 0.05% solution of Tween 20 in PBS. The enzyme reaction was developed with o-phenylediamine dihydrochloride-H
2O
2 in citrate phosphate buffer (pH 5.0), stopped with 8 N H
2SO
4, and recorded at 490 nm by use of a microplate reader (Molecular Devices).
Antibody depletion assay
To determine the relative abundances of antibodies specific for a PfMSP1
19 variant in
P. falciparum-infected human sera, an antibody depletion assay was carried out as described earlier [
25]. The wells of flat-bottom Immunolon-2 plates were coated with 100 ng of each of the PfMSP-1
19 recombinant antigens, namely PfMSP1
19QKNG-L, PfMSP1
19EKNG-L and PfMSP1
19ETSR-F. The wells were blocked with 5% low-fat milk in PBS (pH 7.2) for 1 h at room temperature. After blocking, all of the wells were washed with 0.05% Tween 20 in PBS and then with PBS (pH 7.2), the first two wells in the first column were incubated for ten minutes with
P. falciparum-infected human sera of the respective sequence variants at a dilution of 1:100, while the remaining wells contained only wash buffer. The sera from the first two wells were transferred to the next respective wells in the second column and incubated for half an hour. These serial incubations were carried out until all of the antibodies with respect to a particular antigen were depleted, as determined by color development in the wells by a standard ELISA. It was observed that for all the sequence variantss, antigen-specific antibodies were completely removed from the respective sera after six serial transfers except in the case of Q-KNG-L variant which depleted the sera after nine serial transfers. All these antibody-depleted sera were subsequently analysed for their cross reactivity with each of the PfMSP1
19 variants; PfMSP1
19Q-KNG-L, E-KNG-L and PfMSP1
19E-TSR-F antigens by ELISA. The reactivity of each protein with the other two depleted sera was compared with the undepleted sera to analyse the relative contribution of each antigen.
Discussion
Sequence heterogeneity in PfMSP-1
19 protein, a leading vaccine candidate antigen for vaccination against erythrocytic stages of
P. falciparum, may compromise its use as a vaccine candidate antigen. Based on sequence variations among different laboratory and field isolates, two prototypic alleles of
P. falciparum MSP-1 represented by PfMAD20 and PfK1/Wellcome have been described in isolates from Africa, Asia and Latin America [
15,
16,
24,
25]. The C-terminal 19 kDa region of MSP-1 (PfMSP-1
19) is a highly conserved region showing amino-acid alterations at only five positions out of 102 residues. Based on these alterations, about ten different PfMSP-1
19 allelic forms have been predicted in different field isolates across the world [
19].
Here, sequence diversity in MSP-1
19 region, among Indian
P. falciparum field isolates in malaria endemic villages in same geographical location was investigated. Some of these isolates have been previously analysed for sequence variations in the receptor binding domain of
Pf EBA-175 [
29]. Sequence data of the 16
th block of MSP-1 revealed that all the isolates were of PfMAD20 type. The present study's results and the data obtained from previous studies clearly demonstrate that MAD20 allelic form predominates among Indian PfMSP-1 isolates from different geographical locations [
18,
20,
30]. In the 17
th block, seven allelic forms was observed. Of these PfMSP-1
19 alleles, two alleles (E-TNR-L & E-TSR-F) have not been reported earlier, however, Qari et al (1998) have earlier predicted E-TNR-L. These allelic forms seem to have generated due to intragenic recombination between the two prototypic alleles as suggested earlier because all the substituted amino acids belonged to either one or other allelic form.
To know whether these five amino acid substitutions in the PfMSP-1
19 region generate variant specific immune responses in
P. falciparum infected patients, three PfMSP-1
19 variants (Q-KNG-L, E-KNG-L & E-TSR-F) were expressed in an
E. coli expression system with an N-terminal His tag. Proteins were purified on a Ni
2+-NTA column. All the three PfMSP-1
19 recombinant forms were recognized well by an invasion inhibitory monoclonal antibody (12.10) and anti-PfMSP-1
19 (Q-KNG-L) polyclonal antibody. An earlier study [
28] showed that the yeast secreted recombinant MSP-1
19 variants are recognized by polyclonal antibody raised against baculoproduced MSP1 (E-KNG) variant and by mAB 5B1 that is known to inhibit invasion. Recognition of all the PfMSP-1
19 variants by mAB 5B1 and 12.10 suggest that some of these protective epitopes are not variant specific. A preliminary investigation of protective type IgG1/IgG3 antibody response to Q-KNG-L sequence variant was carried out and significant IgG1/IgG3 responses were observed in the infected sera (data not shown). The frequency of IgG1 responses in different sera was quite similar while a significant difference was seen within IgG3 responses as previously shown [
31].
To determine the presence of variant specific antibodies during the natural infection, sera from BK3-62, CM3-56 and PP3-18 patients were immuno-depleted using each of the three recombinant antigens and the depleted sera were analysed for reactivity with three variant antigens. Results demonstrated that sera from
P. falciparum infected patients do contain cross-reactive as well as PfMSP-1
19 variant specific antibodies. At present, it is difficult to say up to what extent these variant specific antibodies contribute to protective immune responses. Another aspect that requires probing is the contribution of historic infections with parasite of different sequence types. However, data from a number of challenge studies have also provided evidences for the variant specific immune responses. Immunization of mice with recombinant PyMSP-1
19 protected mice against homologous but not heterologous sporozoite or blood stage challenges [
32,
33]. In some cases where some level of protection to heterologous malarial challenge was seen, it was significantly lower than the homologous challenge [
6,
34].
Conclusion
In summary, the present study has shown that PfMAD 20 is the major PfMSP-1 allelic form present in Indian population and a number of PfMSP-119 allelic forms exist even in a single geographical location in a malaria-endemic region in India. The present study further show that significant levels of cross reactive antibodies are generated against different PfMSP-119 allelic forms in a P. falciparum infected natural human population. However, presence of PfMSP-119 allele specific antibodies was also observed in this population thus suggesting, for a better protection against all strains of P. falciparum, it will be appropriate to use a vaccine containing all possible sequence variants. Although only three PfMSP-119 variants were expressed and subsequently analysed for the presence of specific antibodies in the present study, a detailed study is required to clearly understand the role of PfMSP-119 variants in eliciting a protective immune response.
Authors' contributions
AM carried out polymorphism studies, SS expressed the proteins and carried out the ELISAs and depletion assays. Both AM and SS contributed equally in the study. SSD participated in depletion assays, SKS was involved in sample collection, PKT participated in sample collection, TA provided with sera samples, HJ handled sequence data, PM participated in design and coordination of the project along with drafting the manuscript. All authors read and approved the final manuscript.