Nuzhat A. Kaushal

Merozoite surface protein 1 of Plasmodium vivax induces a protective response against Plasmodium cynomolgi challenge in rhesus monkeys.

ABSTRACT The 42-kDa fragment of the merozoite surface protein 1 (MSP-142) is a leading candidate for the development of a vaccine to control malaria. We previously reported a method for the production of Plasmodium vivax MSP-142 (PvMSP-142) as a soluble protein (S. Dutta, L. W. Ware, A. Barbosa, C. F. Ockenhouse, and D. E. Lanar, Infect. Immun. 69:5464-5470, 2001). We report here a process to manufacture the same PvMSP-142 protein but as an insoluble inclusion body-derived protein which was then refolded in vitro. We compared the immunogenicity and protective efficacy of the soluble and refolded forms of PvMSP-142 protein by using a heterologous but closely related P. cynomolgi-rhesus monkey challenge model. As comparative controls we also expressed, purified, and immunized rhesus with the soluble and refolded forms of the P. cynomolgi MSP-142 (PcMSP-142) proteins. All proteins induced equally high-titer, cross-reacting antibodies. Upon challenge with P. cynomolgi, none of the MSP-142-vaccinated groups demonstrated sterile protection or a delay in the prepatent period. However, following an initial rise in parasitemia, all MSP-1-vaccinated animals had significantly lower parasite burdens as indicated by lower cumulative parasitemia, lower peak parasitemia, lower secondary peak parasitemia, and lower average daily parasitemia compared to the adjuvant control group (P < 0.05). Except the soluble PcMSP-142 group, monkeys in all other groups had fewer numbers of days with parasitemia of >10,000 parasites mm−3. Interestingly, there was no significant difference in the level of partial protection observed in the homologous and heterologous groups in this challenge model. The soluble and refolded forms of PcMSP-142 and PvMSP-142 proteins also appeared to have a similar partially protective effect.

Identification of antigenic proteins of setaria cervi by immunoblotting technique.

Identification and characterization of antigenic proteins of Setaria cervi (bovine filarial parasite) adults and microfilariae was done by immunoblotting technique using hyperimmune rabbit sera against S. cervi and Brugia malayi. The antigens recognized by these sera were detected by using 125I protein-A followed by autoradiography. Fifteen different antigens were observed to be common between adult and microfilarial stages of the parasite. Some stage specific antigens were also identified. Many antigens of S. cervi adults and microfilariae were also recognized by rabbit anti-B.malayi serum showing the existence of common antigenic determinants between the bovine and human filarial parasites.

Cloning, overexpression, purification and characterization of Plasmodium knowlesi lactate dehydrogenase.

Plasmodial lactate dehydrogenase, key enzyme of anaerobic glycolysis, has been shown to be a potential immunodiagnostic marker as well as a novel target for chemotherapy. We have cloned, overexpressed and immunochemically characterized the recombinant lactate dehydrogenase of Plasmodium knowlesi, the fifth human malaria parasite. The P. knowlesi lactate dehydrogenase (PkLDH) gene was PCR amplified and 0.9 kb PCR product was cloned into pGEM-T Easy vector. Sequencing and BLAST analysis revealed open reading frame of 316 amino acids of PkLDH showing 96.8% homology with Plasmodium vivax LDH and around 90% with Plasmodium falciparum, Plasmodium malariae and Plasmodium ovale LDHs. The PkLDH gene was subcloned into pGEX-6P1 expression vector and the SDS-PAGE analysis revealed that about 70% of fusion protein was present in the soluble fraction. The fusion protein was cleaved with PreScission protease and recombinant PkLDH (34 kDa) was affinity purified to homogeneity. The purified PkLDH exhibited high reactivity with polyclonal and monoclonal antibodies against plasmodial LDH. The polyclonal antibody produced against purified recombinant PkLDH in rabbits showed high ELISA reactivity with both native and recombinant PkLDH and could detect parasite LDH in malaria infected blood samples by sandwich ELISA. The purified recombinant PkLDH can be used to produce P. knowlesi specific monoclonal antibodies for specific diagnosis of P. knowlesi infection in humans.

Antibodies to lactate dehydrogenase of Plasmodium knowlesi are specific to Plasmodium species.

Polyclonal immune monkey serum raised against schizonts of Plasmodium knowlesi (H-strain) showed the presence of antibodies to lactate dehydrogenase (LDH) of P. knowlesi by immunodot enzyme staining method. The anti-LDH antibodies are most probably directed towards an epitope distinct from the catalytic site as shown by the specific enzyme staining of LDH after binding with antibody on nitrocellulose paper. These antibodies showed reactivity with LDH from different strains (H, P and W1 strains of P. knowlesi) and species (P. cynomolgi B, P. berghei, P. yoelii, P. falciparum and P. vivax) of malarial parasites but did not cross-react with three isoenzymic forms of mammalian LDH (A4, B4 and C4) as well as with LDH from some protozoan and helminth parasites. These findings suggest that the anti-LDH antibodies have defined specificity to Plasmodium spp.

DIAGNOSIS OF MALARIA BY DETECTION OF PLASMODIAL LACTATE DEHYDROGENASE WITH AN IMMUNODOT ENZYME ASSAY

We have previously demonstrated, using polyclonal and monoclonal antibodies, that the lactate dehydrogenase (LDH) of malaria parasites is immunologically distinct from the host enzyme. The polyclonal antibodies, produced against the affinity purified plasmodial LDH (pLDH) in rabbits, showed specificity to LDH of malaria parasites. In the present study, these anti-pLDH polyclonal antibodies were used to develop an immunodiagnostic test (immunodot enzyme assay of plasmodial LDH) based on the detection of parasite LDH in patient blood. The immunodot enzyme assay of plasmodial LDH was evaluated using blood samples from patients with malaria or other infections. Out of 502 microscopically positive malaria blood samples, 497 blood samples showed positive immunodot assays of pLDH while all the 423 microscopically negative cases were found negative by our test. The blood samples from other infections and non-endemic controls were negative by the immunodot enzyme assay of pLDH. This LDH based test was also found negative in blood samples of cured patients 7 days after chloroquine treatment. The test is simple to perform, can be read visually, econimal, highly specific with a sensitivity of ∼99% and is thus suitable for accurate diagnosis of malaria in field conditions.

Protein and Antigenic Composition of Adult and Microfilarial Stages of Setaria Cervi

The protein and antigenic pattern of adult (female/male) and microfilarial stages of Setaria cervi, a bovine filarial parasite, was studied using certain immunochemical techniques. SDS-polyacrylamide gel electrophoretic analysis showed the presence of 35-40 protein bands in adults and 25-29 protein bands in microfilariae in molecular weight range of 10,000-200,000. Immunoelectrophoresis revealed the presence of 9-10 precipitin lines in adult and only 4 precipitin lines in microfilarial antigenic preparations. Crossed immunoelectrophoresis resolved these antigenic preparations further, and revealed the presence of 22-24 antigens in adults and 12-14 in microfilariae. These results demonstrate complex nature of somatic extracts of adult stage as compared to microfilariae and also reveal some qualitative and quantitative differences between these stages.

Isolation of an Antigen Fraction from Setaria cervi Adults Having Potential for Immunodiagnosis of Human Filariasis

Crude antigenic preparations from heterologous filarial parasites gave false positive results because of complex nature of these antigens and their cross-reactivity with other helminth parasites. In the present study, efforts have been made to isolate and characterize the antigens from Setaria cervi important for diagnostic purposes. The fractionation of S. cervi somatic antigenic preparation on Sephacryl S-200 resulted in separation of three major antigenic peak fractions. Crossed immunoelectrophoretic analysis, using immune rabbit serum, revealed 13–14 antigens in SFP-I pool fraction, which showed high reactivity with filarial patients sera as compared to other two pool fractions. This SFP-I fraction was further purified by DEAE-Cellulose column chromatography. Out of the 4 antigen pool fractions, DFP-IV fraction showed high ELISA reactivity with filarial patient serum pool (Wuchereria bancrofti and Brugia malayi) as compared to other fractions. The SDS-PAGE analysis of DFP-IV fraction revealed 2 major and 1 minor protein bands (mol. wt. range 65–70 kDa). Crossed immunoelectrophoresis also showed the presence of 3 antigenic peaks in DFP-IV fraction. The purified DFP-IV fraction showed high reactivity with filarial patients sera but did not cross-react with sera from ascaris and hookworm infections thereby suggesting the filaria-specificity and potential for immunodiagnosis of human filariasis.

Cloning, overexpression and characterization of soluble 42kDa fragment of merozoite surface protein-1 of Plasmodium vivax.

Plasmodium vivax represents the second most prevalent malaria species of major public health importance and the global eradication of malaria requires the development of vaccines to prevent infection. The lack of in vitro culture and a suitable animal model for P. vivax malaria are the major problems for the delay in developing a functional vivax vaccine. A number of antigens have been identified for P. vivax as potential malaria vaccine candidates and among these 42kDa fragment of merozoite surface protein-1 (MSP-142) is one of most promising antigen of asexual blood stage. In most of the earlier studies, the MSP-142 of malaria parasites was expressed as insoluble protein in inclusion bodies and it is difficult to get purified protein in conformation form. In the present study, we have cloned, overexpressed and characterized the 42kDa fragment of P. vivax MSP-1 as soluble protein in Escherichiacoli. The 42kDa gene fragment of P. vivax MSP-1 was PCR amplified using specific primers, sequenced and subcloned into pTriEx-4 expression vector. The optimum expression of recombinant P. vivax protein was obtained in SOC growth medium by inducing with 0.2mM IPTG at 37°C for 4h. The SDS-PAGE analysis showed a fusion protein of 55kDa and about 80% was present in soluble form. The purified P. vivax MSP-142 was characterized and found to be correctly folded and in conformation form as evident by CD spectroscopy, presence of 1 free -SH group and the reactivity with reduction sensitive conformational monoclonals against P. vivax MSP-142.

Production and characterization of monoclonal antibodies against substrate specific loop region of Plasmodium falciparum lactate dehydrogenase.

Plasmodial lactate dehydrogenase, terminal enzyme of the glycolytic pathway, has been shown to be biochemically, immunologically and structurally different from the mammalian enzyme. The substrate specific loop region of plasmodial lactate dehydrogenase (pLDH) has 5 amino acids insert (DKEWN) important for anti-malarial drug targeting. In the present study, we have produced six monoclonal antibodies, which are against three different epitopes of Plasmodium falciparum LDH (PfLDH). Two of these monoclonal antibodies (10C4D5 and 10D3G2) are against the substrate specific loop region of PfLDH (residues 98-109, AGFTKAPGKSDKEWNR). The 10C4D5 and 10D3G2 monoclonals bind to substrate specific loop region resulting in inhibition of PfLDH activity. A Microplate Sandwich ELISA was developed employing high affinity non-inhibitory (10A5H5, Kaff 1.272 ± 0.057 nM) and inhibitory (10C4D5, Kaff 0.306 ± 0.011 nM) monoclonal antibodies and evaluated using gossypol, a well known inhibitor of pLDH. The binding of gossypol to substrate specific loop region resulted in inhibition of binding of 10C4D5 monoclonal. This Microplate Sandwich ELISA can be utilized for identification of compounds inhibitory to PfLDH (binding to substrate specific loop region of parasite LDH) from combinatory chemical libraries or medicinal plants extracts. The Microplate Sandwich ELISA has also shown potential for specific diagnosis of malaria using finger prick blood samples.

Ancylostoma Ceylanicum: I. Protein and Antigenic Composition of Adult and Larval Stages

The protein and antigenic composition of adult and larval stages of Ancylostoma ceylanicum, a human hookworm maintained in golden hamsters (Mesocricetus auratus), was studied employing immunochemical techniques. SDS-polyacrylamide gel electrophoresis revealed the presence of 47 and 43 protein bands in adult worms and infective larvae respectively in the molecular weight range of 10-170 kD. Crossed immunoelectrophoretic analysis, using immune rabbit sera, showed the presence of 32 antigenic peaks in adults and 19 in infective larval stage. Most of the antigens were common between adult and larval stage as evidenced by cross-line immunoelectrophoresis, although some stage specific antigens were also identified. These studies also demonstrate the complex nature of adult worms as compared to larvae.