Molecular cloning and serological characterization of a new Plasmodium vivax recombinant antigen which contains apolipoprotein B-100 like sequences

Vol. 184, No. 2, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

April 30, 1992 Pages 668-672

MOLECULAR CLONING AND SEROLOGICAL CHARACTERIZATION OF A NEW PLAShfUDlUM VZVYXRECOMBINANT ANTIGEN WHICH CONTAINS

APOLIPOPROTEIN B-100 LIKE SEQUENCES

PRATIMA RAY and Y.D. SHARMA*

Department ofBiotechnology, All India Institute of Medical Sciences, New Delhi 110029, India

Received March 17, 1992

SUMMARE We have isolated a new recombinant antigen ‘PV12’ of Plasmodium vivax by immunological screening of the genomic library. The antigen shows a sequence homolo with human apolipoprotein B-100. A large number of P. vivax infected individuals (79% were seropositive against this antigen.

g4;

evade the host defence system The probable function of this antigen could be to

(i) by inactivating the perforin of natural killer cells and (ii) by engaging the host immune system to produce antibodies against this decoy antigen. 0 1992 Academic Press, 1°C.

Plasmodium vivax causes malaria in humans. It is most common of all human malaria parasites, affecting large number of individuals in several tropical countries. The disease remains uncontrolled to-date (42). Progress towards the development of a malaria vaccine for this parasite has been slower compared to Plasmodium falciparum. This is because most of the immunobiologically significant molecules of P.vivax are yet to be characterized. So far only few genes of this parasite have been studied (3-8). The main reason for this slow progress is the non-cultivable nature of this parasite. The applications of recombinant DNA technology however can help in circumventing this problem. There- fore, more molecular biological studies need to be carried out to investigate unto unde- scribed immunobiologically active molecules which could help in controlling the disease. We have recently constructed a P. vivcax genomic library in lambda gtll and screened with the pooled vivax patients’ sera in order to obtain the immunologically active molecules (8). In the present study we describe a clone named PV12 which expresses a recombinant antigen whose sequences are homologous to human apolipoprotein B-100 (Apo B-100) and the expressed antigen is able to evoke the humoral response in large number of P. vivax patients during acute phase of infection.

MATERIALS AND METHODS

Sera: Individuals suffering from fever were attending the malaria clinic at Delhi during the transmission period of 1989 and 1990. These patients were examined for the presence of malarial parasites by light microscopy. Blood samples were collected from P.vivaw positive

*TO whom correspondence should be addressed.

0006-291X/92 $1.50 Copyright 0 1992 by Academic Press. Inc. All rights of reproduction in an? form reserved. 668


individuals. Blood samples collected from healthy personnels working in the laboratory as well as from blood bank of this Institute were used as uninfected healthy controls from India. Blood samples were also collected from European and American (United States) individuals during their visit to the lab. These foreigners had never experienced malaria. We have also collected blood samples from non-parasitemic individuals who had single, two or multiple episodes of vivax malaria during 1983-1991. Isohrion of the recombinant clone: The construction of P.vivax genomic library in lambda gtll has been described earlier (8). This library was immunologically screened with the pooled serum collected from P.vivu.x patients by the described methods (9,lO). Briefly, the pooled serum was depleted of anti-E.coli antibodies and used to screen the library at a 1:150 dilution. The second antibody was alkaline phosphatase conjugated anti-human IgG (Dakopatts, Glostrup, Denmark) and used at 1:750 dilution. Nucleotide sequencing: The insert of the clone PV12 being smaller in size, was directly subcloned into the Ml3 mp18 and mp19 vectors. The dideoxy chain termination method was used for nucleotide sequencing by using the universal Ml3 primers as described earlier (8,10,11). Preparation of recombinant a&ken: The non-recombinant lambda gtll and the recombinant (PV12) phage particles were lysogenized separately in the E.coli host Y1089. The lysogens were grown at 30°C in liquid cultures till midlog phase. The cultures were shifted to 45OC for 20 minutes and then grown at 38’C for 2 hours in the presence of 7 mM iso- prop+B-D-thiogalactopyranoside (IPTG). The cells were harvested and lysed by freez thawing and sonicating as described before (8,lO). The clear lysates were used for enzyme- linked rmmunosorbant assay (ELISA) and SDS-PAGE. The protein concentration in these lysates was determined by Lowry’s method. ELISA: The ELISA procedure was essentially same as described earlier (12). The lysates from recombinant and non-recombinant clones, containing equal amount of protein, were coated into the wells of the 96 flat-bottom microtiter plate. Each well received either 250 or 500 ng protein and plate was incubated overnight at 4’C. After three washings with TBST (50mM Tri-HCl pH 7.5, 150 mM NaCl and 0.005% Tween-20) the wells were blocked with 3% (w/v) defatted and dehydrated milk in the same buffer. The sera samples at two different dilutions (1:250 and 1:500) were used to react with the coated antigen at 37’C for 1 hour. After washing, the wells were exposed to the 1:500 diluted HRP-conjugated rabbit anti-human IgG (Dakopatts, Denmark) at 37’C for 1 hour. Both antibodies (serum and anti-human IgG) were diluted in blocking buffer. The wells were washed and colour was developed by using the substrate 0-phenylene diamine (0.8 mg/mL OPD in O.lM Citrate-Phosphate buffer pH 5.0 containing 0.01% Hydrogen Peroxide). Reaction was terminated by 7% (v/v) Sulphuric acid and the OD was measured at 492 nm in a Titer- tek Multiscan Reader (Flow Lab&vine, Scotland). For each sample, the OD value of non- recombinant lambda gtll was deduced from the test value obtained for recombinant antigen. Nucleotide sequence accession number The PV12 sequence was assigned EMBL accession number X 5368 1 Stutidcal urn@& Students ‘t’ test was used to determine the p values.

RESULTS AND DISCUSSION

We have isolated a genomic clone denoted ‘PV12’ by immunoscreening of the lambda gtll DNA expression library of P.vivux with the pooled, preadsorbed, serum collected from vivax patients. The use of patients’ sera for library screening was to directly identify the parasite molecules which are involved in the human humoral immune response in vivax malaria. The size of the PVlZinsert was about 450 bp on agarose gel. The nucleotide sequence of this clone is shown in Fig. 1A. It contains 50% G + C contents which are slightly higher for malaria parasite genome. But similar higher G + C contents for other P.

viva genes have earlier been reported; PV200 and PV9 contain 47% and 65% G+ C contents respectively (4,8). In general, the malarial genome is A+T-rich; particularly the non-coding regions (13-15). However, the vivax genome contains slightly higher G+ C


A AspGlnProGlyGlnHisSerGluThrLeuValSerThrLysLys*** GATCAGCCTGGGCAACATAGTGAGACTCTTGTTTCTACAAAAAAA TGAAAAATTAGCCAGGTGTG 65

GTGGTATGCGCCTGTAGTCCCAGCTACTTGGGAGTCCGAGGCAGGAGGATTGCTTGACCAGG~ 130

GTTGAAGGCTACAGTGAACAGGGATTGCACCACTGCACTCCAGCCTGGATGGCAGAGTAAGACCC 195

TATCTCT-GATAAAAAGAAACGAAGGGGAGGGAAGGGAAGGGMAGMGGGGAAAGAAA 260

GGGAGGGAAGGAAGGAAGAGAGAAAGAAGGAAAGCAAA 325

GGGAAGGGGAGGGGAAGGAGAGGGGMGGAGAGAGAGC~GGAGAGGGGAGAGGAGGGGAGGAGAGG 390

GAAGGGAAGGGAAACATAGTCTATGGAGTCGMCAGATGGCTCATGGATC 447

B

1' DQPGQHSETLVSTKK ::: . : :

4381 IMALREEYFDPSIVGWTVKYYELEEKIVSLIKNLLVALKDFHSEYIVSASNFTSQLSSQV

F& IA. Nucleotide sequence of PV12 insert determined by dideoxy chain termination method. The deduced amino acid sequence is in frame with B-galactosidase. B. Amino acid sequence homology of PV12 encoded polypeptide (top line) with hiiman Apo B-100 (bottom line). The sequences are shown in a single letter code.

contents than P.falcipamm (13,15). There are some nucleotide repeat units in the PV12 sequence; a pentamer ‘GGGAA’ is repeated several times whereas the lg-mer ‘GAGGGAAGGGAAGGGAAA’ is repeated twice at bases 229-244 and 387-404. The deduced amino acid sequence of PV12 is also shown in Fig. 1A. This sequence is in frame with the B-galactosidase because the clone is expressing a fusion protein. The encoded peptide is small but it is large enough to form an antigenic domain. The protein homology search showed that it shares a significant amount of homology with human Apo B-100 (Fig. 1B). Apolipoproteins of P. vivaw origin are not yet reported. Therefore, the function of such molecules with regard to this parasite remains unknown. However, it can be stated here that Apo B-100 constitutes the major protein part of low density lipoproteins (LDL) in humans. Its potentials as a perforin inhibitor and thus preventing the lysis of non-targetted cells by the cytotoxic T-lymphocytes and natural killer (NK) cells, has recently been described (16). Therefore, it is tempting to speculate that the malaria parasite is synthesizing (and secreting into serum) this homologous molecule to defend itself against host immune system i.e., by inactivating the perforin released by NK cells.

The humoral immune response generated against the PV12 produced recombinant antigen was determined by ELISA in the serum samples collected from currently infected P.vivax patients and those who were non-parasitemic but experienced vivax malaria episodes during 1983-1991 and uninfected healthy controls from India and abroad (Europe and USA). The ELISA results are shown in Table 1. The overall response shown by infected individuals was significantly higher than uninfected healthy controls either from India or abroad (P< 0.001). Majority of the P.vivax patients, 101 of 128 (79%), were found seropositive against this antigen with an average serum titer of 324. This seropositivity rate was 27% (16 of 59) among uninfected healthy controls from India. None of the European and American (United States) serum was found reactive to this antigen. We have also studied this humoral response in 99 individuals who were non-parasitemic at the time of sample collection but had experienced P.vivav malaria either once, twice or many times during

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Table 1 Humoral Response to the Plusmodium vivax Recombinant Antigen ‘PV12’. ELISA was performed on the serum samples collected from actively infected patients (parasitemic) and non parasitemic individuals. In the latter case the individuals had experienced either single, double or multiple episodes of vivax malaria during 1983-1991.

Individuals No of cases Seropositivity Serum titre (%) (Avg.)

Parasitemic

Active Infection 128 78.9

Non parasitemic (Experienced vivax mafaria during 1983-1991)

Single Attack 41 41.46 Two Attacks 44 47.7 Multiple Attacks 14 57.14

Controls

324

152 186 200

Indian 59 27.27 108 US&Europe 9 Zero ND

ND, not determined: Avg, average.

1983-1991. Several of these individuals (49.5%) were responding to this antigen. The seropositivity rates among those who had one, two or multiple episodes were 41.5% (17 of 41), 47.7% (21 of 44) and 57% (8 of 14) respectively. The seropositivity rates and antibody levels among these individuals increased with the number of episodes. But these differenes were statistically non-significant. The seropositivity rates among these individuals were also not significantly different than the Indian controls. Although these values were significantly higher if compared with foreign controls. In conclusion the PV12 antigen evokes the humoral immune response in majority of the patients during the acute phase of P. vivax

infection. And these antibodies could be lasting for a long period of time. The latter possibility, however, is subjected to the confirmation of across-reacting epitopes not being present in other micro-organisms.

The PV12 antigen described here is of interest because it could be playing a dual role for the parasites’ survival against host defence system. Firstly, by mimicking the apolipoprotein B it will be able to inactivate the cytotoxic factor ‘perforin’ which is produced by natural killer cells, thus, protecting the parasite from being killed by these cells. Secondly, this antigen probably also acts as a decoy protein against which antibodies are produced by the host. Therefore, keeping the host immune system busy to produce antibodies to non- sense proteins the parasite is able to evade the real host immune pressure. The host like sequences in the P. vicar are for the first time reported here. Although such reports are available for P. falciparunz (17-19).

ACKNOWLEDGMENTS: We thank Dr. V.P. Sharma, Dr. M.A. Ansari, Dr. T. Adak, Dr. C.R. Pillai of Malaria Research Centre, Delhi, for providing patients’ blood samples and helpful discussions. We also thank Dr. I.Nath, Dr. Ramesh Kumar and Dr. H.K. Prasad for

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suggestions and critical comments on the manuscript. Database search was performed by Mr. R. Dhansekaran. We thank Mr. Jaideep Tewani for typing the manuscri t. This work was supported by the grants from the Department of Biotechnology, Counci P of Scientific and Industrial Research and Indian Council of Medical Research.

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Molecular cloning and serological characterization of a new Plasmodium vivax recombinant antigen which contains apolipoprotein B-100 like sequences