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Biochemical and immunological characterization of E. coli expressed 42 kDa fragment of Plasmodium vivax and P. cynomolgi bastianelli merozoite surface protein-1

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Biochemical and immunological characterization of E. coli expressed 42 kDa fragment of Plasmodium vivax and P. cynomolgi bastianelli merozoite

surface protein-1

Deep C Kaushal1*, Nuzhat A Kaushal2,Atul Narula1, Niraj Kumar1, S K Puri2, Shitij Dutta3 and David E Lanar3

Divisions of Microbiology1 and Parasitology2

Central Drug Research Institute, Post Box 173, Lucknow 226001, India

&

3Department of Immunology, Walter Reed Army Institute of Research Silver Spring, Maryland 20910, USA

Received 14 June 2007; revised 22 October 2007

Plasmodium vivax is one of the most widely distributed human malaria parasites and due to drug-resistant strains, its incidence and prevalence has increased, thus an effective vaccine against the parasites is urgently needed. One of the major constraints in developing P. vivax vaccine is the lack of suitable in vivo models for testing the protective efficacy of the vaccine.

P. vivax and P. cynomolgi bastianelli are the two closely related malaria parasites and share a similar clinical course of infection in their respective hosts. The merozoite surface protein-1 (MSP-1) of these parasites has found to be protective in a wide range of host-parasite systems. P. vivax MSP-1 is synthesized as 200 kDa polypeptide and processed just prior to merozoite release from the erythrocytes into smaller fragments. The C- terminal 42 kDa cleavage product of MSP-1 (MSP-142) is present on the surface of merozoites and a major candidate for blood stage malaria vaccine. In the present study, we have biochemically and immunologically characterized the soluble and refolded 42 kDa fragment of MSP-1 of P. vivax (PvMSP-142) and P. cynomolgi B (PcMSP-142). SDS-PAGE analysis showed that both soluble and refolded E. coli expressed P. vivax and P. cynomolgi B MSP-142 proteins were homogenous in nature. The soluble and refolded MSP-142 antigens of both parasites showed high reactivity with protective monkey sera and conformation-specific monoclonal antibodies against P. cynomolgi B and P. vivax MSP-142 antigens. Immunization of BALB/c mice with these antigens resulted in the production of high titres of cross-reactive antibodies primarily against the conformational epitopes of MSP-142 protein. The immune sera from rhesus monkeys, immunized with soluble and refolded MSP-142 antigens of both parasites also showed high titered cross-reactive antibodies against MSP-142 conformational epitopes. These results suggested that the soluble and refolded forms of E. coli expressed P.

vivax MSP-142 antigens were highly immunogenic and thus a viable candidate for vaccine studies.

Keywords: Plasmodium vivax, Plasmodium cynomolgi, Malaria, Vaccine, Merozoite surface protein-1, Merozoite, Rhesus monkey.

Malaria is caused by the protozoan parasite of genus Plasmodium and is one of the largest public health problems in the world. About 300-660 million people are affected by malaria worldwide. The disease results in the death of 2-3 million people annually, particularly among African children under the age of five, making development of a malaria vaccine a global health priority1.

Most of the work on malaria vaccine development has been done on P. falciparum and relatively little efforts have been made towards developing P. vivax

vaccine2. P. vivax is infrequently fatal and imposes a serious burden of morbidity in malaria endemic areas outside Africa and according to WHO there may be 15-30 million cases of P. vivax annually. Moreover, strains of P. vivax resistant to chloroquine have been identified in many areas and are expected to spread, leading to further increase in the worldwide incidence and prevalence of vivax malaria3-5. Thus, P. vivax continues to impose a major public health burden in endemic areas and necessitates the continued use of chemoprophylaxis6. Some of the major constraints for the development of vaccine against P. vivax malaria are the relapsing nature of P. vivax hepatic stages, lack of in vitro culture and suitable in vivo monkey model system. The in vivo testing of P. vivax vaccine candidates requires highly specialized monkey model

_____________

*Corresponding author

Tel: 522-2612411-18 Ext 4390, 4339 E-mail: deepkaushal@hotmail.com deepkaushal@yahoo.com

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such as Toque and New World monkey species which are available in limited numbers and may not be sufficient to cope with increasing number of P. vivax vaccine candidates7-9. It is, therefore, necessary to compare and down-select vaccine candidate antigens in preclinical studies using alternate animal model before going to the clinical trials in humans.

P. cynomolgi B, a closely related species to P.

vivax, infects rhesus macaques in South-east Asia and its transmission in humans is also reported10. Two parasites share a similar clinical course of infection11, a reticulocyte-specific invasion12, presence of Schuffner’s dots on infected erythrocytes13, and a dormant liver hypnozoite stage that is responsible for a relapsing blood stage infection14. In addition they have similar genomic GC content and rRNA analysis confirming their close taxonomic relatedness15. High homology of major vaccine candidates such as the apical membrane antigen-116, circumsporozoite protein17, erythrocyte-binding protein18 and the 42 kDa fragment of merozoite surface protein-1 (MSP- 142)19 have been reported. Earlier, P. cynomolgi- rhesus monkey model system has been used to test the efficacy of P. vivax recombinant antigens20,21.

The MSP-1 found on the surface of Plasmodium merozoites has been a prime vaccine candidate22. The 200 kDa precursor molecule of MSP-1 undergoes step-wise proteolytic processing, resulting in 42 kDa protein (MSP-142) on the surface of free merozoites23. This 42 kDa intermediate product undergoes secondary processing at the time of invasion, releasing a 33 kDa soluble polypeptide (MSP-133) and leaving behind a glycosyl-phosphatidylinositol- anchored 19 kDa product (MSP-119) on the invading merozoite23. Depending on species, MSP-119 contains 10 or 11 cysteine residues that form five disulfide bonds. Immunization with recombinant MSP-142 and MSP-119 raise antibodies that inhibit invasion of merozoites and protect monkeys24.

Earlier21, we have evaluated the P. cynomolgi B - rhesus monkey model system for testing the protective potential of E. coli expressed MSP-142

recombinant antigens of P. vivax and P. cynomolgi B and found significant reduction in parasite burden of monkeys immunized with MSP-142 antigens of both parasites21. In the present study, we have biochemically and immunologically characterized the soluble and refolded forms of E. coli expressed MSP- 142 antigens of both parasites using polyclonal and monoclonal antibodies against protective

conformational epitopes and have shown that in vitro folded MSP-142 antigens of these parasites are correctly folded. In addition we have tested the immunogenicity of soluble and refolded MSP-142

antigens of both parasites in mice using Montanide ISA720 an adjuvant suitable for human use.

Materials and Methods

Recombinant MSP-142 proteins and their denaturation, reduction and alkylation

The expression and purification of P. vivax MSP- 142 and P. cynomolgi B MSP-142 soluble (PvS and PcS) and refolded (PvR and PcR) proteins were done as descried earlier21. The recombinant antigens were denatured and reduced by disturbing the disulphide bonds23. The soluble and refolded proteins from both parasites were either alkylated or denatured, reduced and alkylated. Briefly, dithiothretol (DTT, 10 mM) and guanidine hydrochloride (6 M) were added to the recombinant MSP-142 proteins (100 µg/ ml) and incubated at 50oC for 1 h. Thereafter, the denatured and reduced proteins were alkylated by adding sodium iodoacetate (100 mM) and further incubated in dark at 37oC for 1 h. The alkylated or denatured, reduced and alkylated proteins were dialyzed overnight at 4oC and kept at -20oC until used.

SDS-Polyacrylamide gel electrophoresis

The purity of recombinant MSP-142 antigens was judged by SDS-PAGE using 10% acrylamide gels run under non-reducing conditions following the procedure of Lammeli25.

Immunization of mice

BALB/c mice (6-8 weeks old) were immunized with MSP-142 soluble and refolded antigens of both parasites. Institutional Animal Ethics Committee of CDRI approved the immunization protocols. The mice were kept on standard diet in animal house facility of our institute and divided into five groups (Group 1 to 5) with each group comprised 10 mice (5 males and 5 females). Briefly, mice in group 1 were immunized with P. vivax soluble (PvS), group 2 with P. vivax refolded (PvR), group 3 with P. cynomolgi B soluble (PcS) and group 4 with P. cynomolgi B refolded (PcR) antigens. Each mouse was immunized subcutaneously with 0.2 ml of antigen formulation containing 20 µg of antigen in 60 µl and 140 µl of Montanide ISA720 adjuvant (Sppic Inc., France). The mice in group 5 were used as controls and received PBS and Montanide ISA720 adjuvant in the same

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ratio as for mice of group 1 to 4. The mice were given three injections at 15 days interval and were bled 1 week after each injection. The sera collected were kept frozen at -20oC.

Immune monkey sera

Immunization of monkeys with MSP-142 (PvS, PvR, PcS and PcR) was done as described previously21. The monkeys were immunized at four weeks intervals and were bled every two weeks for collection of serum. The monkeys in all the five groups were challenged with P. cynomolgi B blood stage parasites 4 weeks after the last injection and the number of parasites was counted as described elsewhere21. The parasitaemia was expressed as parasites/mm3. After 45 days, the monkeys were cured with chloroquine26.

ELISA

Enzyme-linked immunosorbent assay (ELISA) was done in 96-well plates essentially as described before27,28. Briefly, the wells of microtitre plates were coated with recombinant Pv or PcMSP-142 antigens (40 ng/100 µl/well, diluted in PBS) by incubating the plates at 37oC for 1 h and then overnight at 4oC. The plates were blocked by incubation with 300 µl of 3% non-fat dry milk powder (w/v, prepared in PBS) at 37oC for 2 h. After three washes with PBS-Tween (PBS-T, PBS with 0.05% Tween-20), the plates were incubated with 100 µl of appropriately diluted antibodies (diluted in 1% milk) for 2 h at 37oC, washed again thrice with PBS-T and incubated for 1.5 h with optimally diluted enzyme labeled secondary antibody (horseradish peroxidase-labelled anti-mouse or anti-monkey IgG;

Sigma, USA). After washing the plates with PBS-T, the color was developed by adding the substrate solution (1 mg/ml, o-phenylenediamine in citrate- phosphate buffer, pH 5.0, containing 1 µl/ml H2O2).

The reaction was stopped after 10 min by adding 5 N H2SO4 and the absorbance was read at 490 nm using Molecular Devices UVmax microplate ELISA reader.

Isotypic analysis

The immune mice sera from different groups of mice were tested for IgG, IgG1, IgG2a, IgG2b, IgG3, IgM and IgA antibodies against MSP-142 antigens by ELISA using purified class-specific heavy chain reagents (goat anti-mouse IgM, IgG, IgG1, IgG2a, IgG2b, IgG3, and IgA) and rabbit anti-goat Ig peroxidase conjugate (Sigma, USA).

Results

SDS-PAGE analysis of P. vivax MSP-142 and P. cynomolgi B MSP-142 antigens

The SDS-PAGE pattern of recombinant P. vivax MSP-142 soluble (PvS), refolded (PvR) and P. cynomolgi B MSP-142 soluble (PcS), refolded (PcR) antigens under non-reducing conditions is shown in Fig. 1. All the four recombinant antigens showed a single protein band. The soluble and refolded proteins of both parasites exhibited same mobility on native SDS-PAGE. The SDS-PAGE pattern of soluble and refolded proteins from both parasites before and after alkylation is shown in Fig. 2. The molecular masses of alkylated PvS and PvR of P. vivax (Fig. 2a, lanes 2 and 5) are same and showed slight increment as compared to native proteins (Fig 2a lanes 1 and 4). The reduction and alkylation of PvS resulted in increase in size and thus decreased mobility (Fig. 2a, lane 3). The mobility of reduced and alkylated PvR was the same as reduced and alkylated PcS (data not shown). Similar results were obtained with P. cynomolgi B MSP-142 PcS and PcR proteins (Fig. 2b).

Fig. 1—Coomassie blue stained purified P. vivax soluble (Lane 1), P. vivax refolded (Lane 2), P. cynomolgi B soluble (Lane 3) and P. cynomolgi B refolded (Lane 4) MSP-142 proteins on non- reducing 10% SDS-polyacrylamide gel

Fig. 2—Coomassie blue stained non-reducing 10% SDS- polyacrylamide gel of purified PvMSP-142 (a) and PcMSP-142 (b) proteins [Lane1, soluble; lane 2, soluble alkylated; lane 3, denatured, reduced and alkylated; lane 4, refolded; and lane 5, refolded alkylated]

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The presence of reduction sensitive epitopes was confirmed by reactivity of these antigens with protective monkey sera and conformation-specific monoclonal antibodies against P. vivax and P.

cynomolgi B MSP-142 antigens. The polyclonal (monkey anti-P. cynomolgi B schizont)29 and monoclonal antibodies (Moab1, 2, 3, and 4) showed same level of reactivity (ELISA OD around 3.00) with soluble and refolded MSP-142 antigens of both parasites (Table 1).

Antibody response in immunized mice

The sera obtained from immunized mice were tested in ELISA against MSP-142 antigens of both parasites to measure the levels of antibodies. The sera from five groups (anti-PvS, anti-PvR, anti-PcS, anti- PcR and control group) collected after each injection, were tested in ELISA against both MSP-142 antigens.

The antibody titres of immune mice sera of different bleeds (1st, 2nd, 3rd) from four groups are given in Table 2. All the mice sera (Group 1 to 4) showed increase in antibody titres with number of injections.

Higher antibody titres were obtained with the homologous antigens than heterologous. The sera after 3rd injection showed 6-8 fold and 5-6 fold increase in antibody titres with the homologous and heterologous antigen, respectively. The antibody titres (geometric mean ± 2 SD) observed with sera from groups 1-4 (anti-PvS, anti-PvR, anti-PcS and anti-PcR respectively) with PvS antigen were 207937 ± 1953, 194012 ± 2046, 129516 ± 1708 and 125843 ± 1765.

On using PcS antigen, group 1-4 (anti-PvS, anti-PvR, anti-PcS and anti-PcR respectively) sera showed antibody titres of 142120 ± 1760, 136474 ± 1689, 234833 ± 1320 and 219106 ± 1592. The sera from control (Group 5) did not show any reactivity with any of the PvS and PcS antigens (data not shown).

Reactivity of immune mice serum pools with soluble, refolded and denatured antigens

The reactivities of serum pools from different groups (Group 1–4) of mice were tested with soluble, refolded and denatured MSP-142 antigens of both parasites in ELISA. Serum pools (anti-PvS, anti-PvR,

Fig. 3—Antibody titres of immune mice sera pools of different groups with soluble (S), refolded (R) and denatured (D) MSP-142 antigens of P. vivax and P. cynomolgi B in ELISA

Table 1—Reactivities of soluble, refolded MSP-142 antigens of P.

vivax and P. cynomolgi B with polyclonal and monoclonal antibodies in ELISA

[PvS, P. vivax soluble; PvR, P. vivax refolded; PcS, P. cynomolgi B soluble; PcR, P. cynomolgi B refolded; Moabs 1 & 2 are against the conformation epitopes of P. cynomolgi B MSP-1 and Moabs 3 & 4 are against the conformation epitopes of P. vivax MSP-1 (Kaushal and Kaushal, unpublished). Culture supernatants of these Moabs were used. Immune serum from rhesus monkey, immunized with P. cynomolgi B schizont antigen29, used at 1: 1000 dilution]

ELISA O.D. at 490 nm Antibodies

PvS PvR PcS PcR Immune monkey serum 3.134 3.092 3.427 3.325

MoAb1 2.827 2.735 2.979 2.884

MoAb2 2.924 2.979 3.089 3.125

MoAb3 3.091 3.123 2.865 2.872

MoAb4 3.289 3.186 2.927 2.905

Table 2—Reciprocal antibody titres of serum pools of different bleeds from four groups of mice with P. vivax and P. cynomolgi B MSP- 142 antigens

[Groups 1, 2, 3 and 4 were immunized with P. vivax soluble (PvS), P. vivax refolded (PvR), P. cynomolgi B soluble (PcS) and P.

cynomolgi B refolded (PcR) MSP142 antigens respectively. Group 5 was control group and immunized with adjuvant only. Each group comprised of 10 mice. Antibody titres were expressed as Geometric mean titre ± 2 SD. 1st, 2nd and 3rd were serum bleeds after different injections]

Antibody titres × 1000

PvS MSP142 Antigen PcS MSP142 Antigen

Groups

1st 2nd 3rd 1st 2nd 3rd

Group 1 (PvS) 26.927 ± 1.346 91.106 ± 1.512 207.937 ± 1.953 24.411 ± 1.464 87.486 ± 1.607 142.120 ± 1.760 Group 2 (PvR) 34.703 ± 1.997 93.765 ± 1.594 194.012 ± 2.046 25.124 ± 1.518 80.671 ± 1.677 136.474 ± 1.689 Group 3 (PcS) 26.163 ± 1.072 72.278 ± 1.479 129.516 ± 1.708 32.379 ± 1.708 100.495 ± 1.518 234.833 ± 1.320 Group 4 (PcR) 25.421 ± 1.275 69.406 ± 1.408 125.843 ± 1.765 29.354 ± 1.320 102.217 ± 1.749 219.106 ± 1.592

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anti-PcS, anti-PcR) from different groups of immunized mice showed significantly high reactivity with both soluble and refolded (antibody titres 48,000-128,000 and 32,000-128,000 respectively) MSP-142 antigens of both parasites, while very little reactivity (< 20%) was observed with denatured antigens of these parasites (Fig. 3).

Isotype analysis of immune mice sera

The reciprocal antibody titres for each group of mice with PvS and PcS antigens are shown in Fig. 4a and b. All the four groups (PvS, PvR, PcS, PcR) showed high antibody titres for the total IgG and among isotypes of antibodies, IgG1 titre being the highest, followed by IgG2b, IgG2a and IgM. No significant IgG3 and IgA reactivity was observed with both antigens.

Antibody responses and parasitaemia in immunized monkeys

The serum pools from five groups of monkeys immunized with PvS, PvR, PcS, PcR (Groups 1 to 4 respectively) and Montanide ISA720 adjuvant (Group 5) were tested against PvS and PcS antigens in ELISA and results are given in Table 3. Almost same antibody titres were observed for group 1 and 2 (anti- PcS and anti-PcR) monkey sera with both PvS and PvR antigens. The group 3 and 4 (anti-PcS and anti- PcR) monkey sera showed high antibody titres with Pcs antigen. The titration curve of serum pools from different groups of monkeys (Groups 1 to 4) with PvS and PcS antigens are shown in Fig. 5. Comparable titration curves were obtained with serum pools from all the four groups with both PvS and PcS antigens.

The serum pool from group 5 did not show any reactivity (data not shown).

Fig. 4—Antibody titres of different isotypes in immune mice sera pools of different groups with P. vivax MSP-142 and P. cynomolgi B MSP-142 antigens in ELISA

Table 3−ELISA titres and course of infection in different groups of rhesus monkeys after challenge with P. cynomolgi B parasites [Each group comprised six monkeys. ELISA titres were expressed as Geometric mean titre ± 2 SD and parasitaemia as mean ± SD of number of parasite/mm3. Statistical analysis of parasitaemia data was done by one-way-analysis of variance, followed by Newman Keul’s test for multiple comparisons. Data in parentheses represents the range of lowest and highest parasite numbers in a group of six monkeys.

Daily average parasitaemia was the mean of 35 days parasitaemia. Detailed data on protective potential of P. vivax and P. cynomolgi B MSP142 antigens in P. cynomolgi B rhesus monkey model system was given in our previous publication21]

ELISA Titre × 1000 Parasiatemia

Groups

PvS PcS Primary peak Secondary peak Daily Average

Group 1 (PvS)

111.431 ± 1.859 97.006 ± 2.137 31309.6 ± 21755.9 (12702-56100)

16034.6 ± 11440.1 (5785-34532)

5248.8 ± 3334.0 (2381-9366) Group 2 (PvR)

114.035 ± 1.761 101.593 ± 2.046 48963.5 ± 11349.1 (31239-61040)

29639.5 ± 11695.0 (15132-49824)

9584.2 ± 2116.5 (6792-1252) Group 3 (PcS)

86.272 ± 1.988 143.675 ± 2.839 50640.5 ± 16286.3 (20025-65410)

31483.7 ± 14084.1 (12729-50799)

10068.0 ± 3001.9 (4632-12719) Group 4 (PcR)

80.635 ± 2.046 114.035 ± 1.761 37111.5 ± 18099.7 (15288-55472)

24608.7 ± 13969.4 (3913-41185)

7659.3 ± 3530.6 (3640-11400) Group 5 (Control)

0 0 120124.7 ± 33081.9

(86050-156400)

74779.8 ± 33688.6 (42284-118125)

20560.3 ± 8180.1 (14198-31476)

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The reactivities of PvS, PvR, PvD, PcS, PcR and PcD MSP-142 antigens were tested with immune monkey serum pools (taken 1 week before challenge) from four groups of immunized monkeys (Groups 1-4) in ELISA and the antibody titres are shown in Fig. 6. The denaturation of MSP-142 antigens resulted in significant reduction of ELISA reactivity with immune monkey serum pools. The reciprocal antibody titres of 2000-4000 were obtained with denatured MSP-142 antigens of both parasites. High antibody titres (64,000-128,000) were observed with soluble and refolded antigens (PvS, PvR, PcS and PcR). These results suggested that most of the antibodies in these immune sera (produced by immunization with recombinant MSP-142 antigen) may be directed against the disulphide bonded conformational epitopes of MSP-142 antigens of both parasites.

The parasitaemia data on immunized monkeys (challenged with P. cynomolgi B blood stage

parasites) in all the five groups are given in Table 3.

The parasitaemia of different groups was compared in terms of primary and secondary peaks parasitaemia, and average daily parasiatemia. All the four vaccinated groups showed significantly lower parasite burden (p<0.05) as compared to the control group. No significant difference was observed between the groups immunized with the soluble and refolded proteins (p>0.05).

Discussion

The MSP-1 expressed on the surface of invasive merozoites of Plasmodium is one of the important targets for development of an effective malaria vaccine.

Thus correct folding, purity and yield of recombinant antigen are important criteria for developing MSP-1 malaria vaccine30. The MSP-119 and MSP-142 fragments of MSP-1 are well characterized in P. falciparum31-34, P. cynomolgi7 and P. yoelii35 and are shown to be the targets of protective immunity. Different expression systems have been evaluated for development of recombinant MSP-1 malaria vaccine. These include production of MSP-1 in Saccharomyces cerevisiae36, Pichia pastoris37, baculovirus-infected insect cells34 and milk of transgenic mice38. The E. coli protein expression system, the first commercialized system for recombinant protein is cost-effective and very efficient for non-glycosylated proteins, such as MSP-142. In our earlier study, we have evaluated protective potential of P. vivax and P. cynomolgi B MSP-142 recombinant antigens in P. cynomolgi B- rhesus monkey model system21. In the present study, we have done the biochemical and immunological characterization of

Fig. 5—Titration curves of immune monkey sera pools from different groups with P. vivax MSP-142 (a) and P. cynomolgi B MSP-142 (b) antigens in ELISA [Sera were collected 1 week before challenge]

Fig. 6—Antibody titres of monkey sera pools of different groups with soluble (S), refolded (R) and denatured (D) MSP-142 antigens of P. vivax and P. cynomolgi B in ELISA

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E. coli expressed P. vivax and P. cynomolgi B MSP- 142 antigens and demonstrated that the refolded antigens are homogeneous, correctly folded and immunogenic in nature.

[

SDS-PAGE analysis of recombinant MSP-142

antigens under non-reducing conditions showed a single protein band in the recombinant antigens (PvS, PvR, PcS and PcR). Both the soluble and refolded proteins of P. vivax (PvS, PvR) and P.

cynomolgi B (PcS, PcR) showed the same mobility on native SDS-PAGE. As MSP-142 proteins of both parasites contain odd number of cysteine residues (11), it is likely that one cysteine is not involved in the formation of disulfide bond. In order to test whether refolded protein also has one cysteine free, both soluble and refolded proteins from both the parasites were alkylated and analyzed on SDS- PAGE under non-reducing conditions. The alkylated P. vivax (PvS, PvR) and P. cynomolgi B (PcS, PcR) MSP-142 proteins revealed same molecular mass and showed slight increment in the molecular mass as compared to native proteins thereby suggesting that these proteins were correctly folded. This was further evidenced by the same level of reactivity of refolded and soluble proteins of both parasites with conformation-specific monoclonals (Kaushal and Kaushal, unpublished observation) and protective polyclonal antibodies29. The reduction and alkylation of PvS, PvR, PcS and PcR resulted in significant increase in molecular mass and thus decrease in mobility. These results suggest that reduction may result in breakage of disulfide bonds and make the sites available for alkylation

Higher antibody titres were obtained in sera from four groups of mice (anti-PvS, anti-PvR, anti-PcS, and anti-PcR) with soluble and refolded MSP-142

antigens of both parasites suggesting that these antigens are highly immunogenic in nature. Isotype analysis revealed a predominance of IgG1 antibody in immune mice sera. Earlier similar observations for predominance of IgG1 responses were reported in mice immunized with P. yoelii MSP-119

39 and P.

vivax MSP-119

40 as well as in P. vivax-infected patient sera40. High reactivity of serum pools from different groups of immunized mice with both soluble and refolded antigens of both parasites and very little reactivity with denatured antigens suggested that the antibodies in immune mice sera were directed against the conformational epitopes.

The protective response to MSP-1 is shown to be antibody-mediated in in vitro invasion inhibition assay and passive transfer experiments41. Monoclonal and polyclonal antibodies against several antigens of P. cynomolgi and P. vivax are found to be cross-reactive42,43 and P. cynomolgi sporozoites protect humans against a P. vivax challenge44. In our earlier study21, because of close phylogenic relationship between P. cynomolgi and P.

vivax, we used P. cynomolgi B rhesus monkey model to compare the immunogenicity and efficacy of soluble and refolded P. vivax MSP-142 products. We found that the soluble and refolded PvMSP-142

products showed similar immunogenicity and efficacy. Immunized monkeys in both the homologous and heterologous challenge groups showed similar reduction in parasite burden as compared to the adjuvant control groups21.

The Montanide ISA720 adjuvant has been used in several human vaccine trials45 and in combination with PvMSP-142 induces both B and T cell responses in mice40,46. In the present study, immunization of mice with P. vivax and P. cynomolgi B MSP-142

recombinant antigens adjuvanted with Montanide ISA720 induced high level of MSP-1 specific antibodies. In our earlier study, vaccination of rhesus monkeys with these antigens in combination with Montanide ISA720 induced a partially protective immune response in primates using an adjuvant acceptable for human use21. Similarly, partial protection has been reported in vaccine trials using PvMSP-119 adjuvanted with a non-ionic block copolymer in the splenectomized Saimiri monkey model against P. vivax challenge8,47. Baculovirus produced MSP-142 and MSP-119 fragments of P.

cynomolgi induce a high degree of sterile protection;

however, Freund’s complete adjuvant (not suitable for human use) was employed7.

Conclusion

In the present study, we have biochemically and immunologically characterized the soluble and refolded P. vivax and P. cynomolgi B MSP-142

antigens expressed in E. coli. SDS-PAGE analysis shows that both soluble and refolded E. coli expressed MSP-142 antigens of both parasites are homogenous in nature. The reactivities of MSP-142 refolded antigens with antibodies against conformational epitopes suggest that these proteins are correctly refolded.

Immunization of mice with MSP-142 antigens of both

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parasites induced high titres of antibodies primarily against the conformational epitopes. These findings suggest that E. coli expressed soluble and in vitro refolded P. vivax MSP-142 antigens are immunogenic in nature and can be used for vaccine studies.

Acknowledgements

This study was funded by grants no. 990352 and no. 990339 from the WHO to DCK and DEL. We would like to thank Dr. C M Gupta, Director, CDRI for his support for this work. One of the authors (NK) is grateful to the Council of Scientific and Industrial Research for Junior Research fellowship (CDRI Communication no.7262).

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References

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