INFECTION AND IMMUNITY, May 1990, p. 1408-1414 0019-9567/90/051408-07$02.00/0 Copyright © 1990, American Society for Microbiology

Vol. 58, No. S

Human Immune Response Directed against Plasmodium falciparum Heat Shock-Related Proteins KUMAR,'*

YING ZHAO,1 PATRICIA GRAVES,2 JORGE PEREZ FOLGAR,lt LEE MALOY,3t AND HONG ZHENG' Department of Immunology and Infectious Diseases, School of Hygiene and Public Health, Johns Hopkins University, NIRBHAY

Baltimore, Maryland 212051; Queensland Institute for Medical Research, Brisbane, Australia2; and National Institutes of Health, Bethesda, Maryland 208923 Received 1 November 1989/Accepted 9 February 1990

Heat shock-related stress proteins present in all eucaryotes and procaryotes have been shown to be immune a broad range of infections. We have analyzed sera from people exposed primarily to Plasmodium falciparum for specific antibodies against two heat shock-related proteins (proteins similar to the heat shock protein with a molecular weight of 75,000 [Pfhsp] and a glucose-regulated protein with a molecular weight of 72,000 [PfgrpJ). In an immunoprecipitation analysis with metabolically labeled parasites and synthetic peptides in an enzyme-linked immunosorbent assay, specific antibodies against Pfhsp and Pfgrp were detected in the sera of these individuals. Sera from people exposed to a different human malarial parasite, Plasmodium vivax, did not react with the peptides in an enzyme-linked immunosorbent assay. Southern blot analysis with DNA isolated from P. falciparum from different geographical locations showed a conservation of genes for these stress proteins; thus, they are likely to be immune targets in various endemic areas. Lymphocytes from two tested immune donors responded in proliferation assays to purified Pfhsp and Pfgrp and purified recombinant proteins. However, a similar response was also seen in lymphocytes from nonimmune individuals and has raised questions pertaining to a generalized responsiveness of lymphocytes to some common determinants present in heat shock-related proteins in various pathogens. targets in

Heat shock proteins (hsps) (stress proteins) have recently gained prominence in the field of infectious diseases. Heat shock-related protein antigens have been identified, and genes have been cloned in various pathogens including the protozoan parasite Plasmodium falciparum (2, 3, 10, 21, 27), Leishmania species, Trypanosoma species, nematode Brugia malayi, trematode Schistosoma spp. (see reference 16 for a review), Salmonella typhimurium, Coxiella burnetii, Legionella species, Treponema species, Borrelia species, and Mycobacterium species (see reference 29 for a review), raising questions pertaining to their role during infection. They are the major targets of immunity (both antibody and T-cell responses) against Mycobacterium tuberculosis and Mycobacterium leprae (18, 19, 28, 30). Recently, it has also been shown that y5' T cells accumulate during infection and epitopes in the 65-kilodalton (kDa) hsp of Mycobacterium species stimulate T cells expressing -yi receptors (9, 15, 17). Heat shock-related proteins include proteins commonly known as either hsps (hsp7O, hsp90, hsp20, etc.) or glucoseregulated proteins (grp78 or grp94). The precise functions of these heat shock-related stress proteins are not known. However, it has been suggested that they play a role in cellular growth and development, thermal adaptation, protein folding, and translocation (4, 5, 12, 20). The synthesis of hsps is induced not only by elevation in temperature but also by various physiological and nonphysiological stimuli, e.g., glucose deprivation, heavy metals, inhibitors of energy metabolism, perturbation of glycosylation and calcium homeostasis, oxidant stress, and ethanol (12, 20).

Malarial parasites, like other parasites, spend parts of their life cycles in warm-blooded vertebrate hosts and the remainder in poikilothermic invertebrate vectors. Host responses to infection include inflammation and fever resulting in an elevation in temperature either locally or systemically. While immune mediators such as interleukin-1, interleukin2, tumor necrosis factor (TNF; cachectin), and interferons are considered to be endogenous pyrogens, there exists a strong link between immune mediators and heat shock responses (8, 22, 25). More recent studies have shown that two genes for hsp70 map within the region of major histocompatibility complex (24). Elevation in the temperature of the environment of the parasites leads to induction of various heat shock-related proteins in P. falciparum (N. Kumar et al., submitted for publication). As the major cellular proteins, their release either by leakage or during parasite death during fever might make them available for presentation to various effector cells of the immune system. There are a number of examples in support of stress proteins being major immune targets during infections, especially in Mycobacterium species. We have studied immune responses against malarial stress proteins during natural infection and report here that some epitopes in the hsps are highly immunodominant and also contain determinants for stimulation of T lymphocytes. (This work was presented at the 38th Annual Meeting of the American Society of Tropical Medicine and Hygiene, Honolulu, Hawaii, December 1989.) MATERIALS AND METHODS Parasites. P. falciparum parasites (clones and isolates) were maintained in culture as described by Trager and Jensen (26). Briefly, parasites were cultured in RPMI 1640 medium containing 10% normal human sera (NHS)-0.37 mM hypoxanthine-29 mM sodium bicarbonate in the presence of

Corresponding author. t Present address: Universidad de San Carlos de Guatemala, Escuela de Quimica Biologia, Guatemala, C.A. t Present address: Magainin Sciences, 550 Pinetown Road, Fort Washington, PA 19034. *

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IMMUNE RESPONSE TO MALARIAL HEAT SHOCK PROTEINS

normal human erythrocytes (5% hematocrit) at 37°C in a gaseous environment of 3% 02, 3% CO2, and balance N2. The medium was changed daily, and parasite growth was monitored by microscopic examination of methanol-fixed, Giemsa-stained smears. Gametes used in the cell proliferation assays were purified from culture-derived gametocytes of P. falciparum by the method described in detail elsewhere

(23).

Sera. Sera were isolated from venous blood and shipped frozen. Sera were obtained from people (ages, 14 to 55 years) living in the malaria-endemic area Agan (Madang province) in Papua New Guinea and from people (ages, 5 to 64 years) living in Plasmodium vivax-endemic areas in Guatemala. The crude parasite rate at the time of blood collection in Papua New Guinea was 30% (21.4% for P. falciparum, 1.4% for P. vivax, and 8.6% for Plasmodium malariae) (7). At the time of blood collection, only four donors and one donor were blood smear positive for P. falciparum and P. malariae, respectively. All the donors in Guatemala were positive for the presence of P. vivax at the time of blood collection. Antigens and peptides. The cloning of genes for P. falciparum hsp with a molecular weight of 75,000 (Pfhsp) and a glucose-regulated protein with a molecular weight of 72,000 (Pfgrp) from the lambda gtll expression library and the preparation of lysogens have been described previously (10). Various recombinant proteins were purified from the lysates of lysogens expressing Pfhsp or Pfgrp as fusion proteins or wild-type lambda gtll. Purification was achieved by affinity chromatography with rabbit anti-p-galactosidase coupled with Sepharose beads (Organon Teknika) and electroelution. Briefly, lysates were treated with 100 p,g of lysozyme (Sigma Chemical Co.) per ml and 10 ,ug of DNasel (Boehringer Mannheim Biochemicals) per ml for 30 min at room temperature and made up to 0.05% 3-[(3-chloamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS)-0.5% Triton X100-0.5% sodium dodecyl sulfate (SDS) and further incubated for 60 min on ice. Lysates were clarified by centrifugation (10,000 x g, 4°C, 20 min), and supernatant was recirculated through the antibody column for 3 to 4 h at room temperature or overnight at 4°C. The column was washed with 10 volumes of borate-buffered saline (0.2 M boric acid, 0.16 M NaCl, pH 8.0), and bound proteins were eluted with 0.1 M Na2CO3-NaHCO3, pH 10.8. Fractions were analyzed for the purity and location of the fusion proteins. Appropriate fractions were concentrated by SpeedVac (Savant Instruments, Inc.). Affinity-purified proteins were further fractionated by SDS-polyacrylamide gel electrophoresis (PAGE), and the fusion proteins were recovered from the gel slices by electroelution with the Bio-Rad electroeluter. Samples were dialyzed against RPMI 1640, protein concentration was measured by the BCA (Pierce Chemical Co.) method, and the samples were sterilized and stored at -20°C. Pfhsp and Pfgrp were also purified directly from the parasites. Parasitized erythrocytes were separated from uninfected erythrocytes by centrifugation through 50% PERCOLL (23) and extracted in SDS buffer for SDS-PAGE. Proteins in the gels were electroblotted onto nitrocellulose membranes, and regions containing Pfhsp or Pfgrp were identified by immunoblotting with rabbit antisera prepared against fusion proteins (10). Nitrocellulose membrane pieces (5 by 15 mm) were solubilized in 500 ,lI of dimethyl sulfoxide (rocking, >1 h), and precipitated by very slow addition of 0.05 M carbonate buffer (pH 9.6) while being vortexed vigorously (1). The precipitate thus obtained was washed three times with RPMI 1640 and finally suspended in 1.0 ml of RPMI 1640. Large pieces were removed by letting the

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suspension stand for 10 s, and the suspension containing microparticles was stored at -20°C and used as a source of parasite-purified proteins. Peptides corresponding to unique sequences GGMPGGM PGGMPGGMNFP for Pfhsp and SGDEDVDSDEL for Pfgrp (10) were synthesized by solid-phase peptide synthesis chemistry and further purified by high-pressure liquid chromatography. Metabolic labeling, immunoprecipitation, SDS-PAGE, and fluorography. Cultured parasites at 10 to 15% parasitemia were washed three times in cysteine- and methionine-free RPMI 1640. Washed cells were resuspended in the same medium and incubated in the presence of 50 ,uCi of trans[35S] label (specific activity, .1,005 Ci/mmol; ICN Biomedicals Inc.) per ml for 2 h at 37°C. Labeled cells were washed three times with regular RPMI 1640 and solubilized in the extraction buffer containing 10 mM Tris-150 mM NaCl-1% Triton X-100-2 mM phenylmethylsulfonyl fluoride (Sigma)0.2 mM L-1-tosylamide-2-phenylethylchloromethyl ketone (Sigma)-0.2 mM p-tosyl-L-lysine chloromethyl ketone (Sigma)-10 mM EDTA-0.25 ,ug of leupeptin (Boehringer Mannheim) per ml-10 ,ug of pepstatin A (Sigma) per ml at 4°C for 1 h. Extracts were centrifuged for 30 min at 15,000 x g at 4°C, and the supernatants were used in the immunoprecipitation analysis. Extract corresponding to 100,000 trichloroacetic acid-insoluble cpm were incubated with various sera (10 ,ul) in a final volume of 150 pR1 overnight at 4°C. Protein A-Sepharose (Pharmacia Fine Chemicals; 100 pjl of a 25% suspension) was added to each tube and incubated further for 1 h at room temperature on a rocker to adsorb antigenantibody complexes. Beads were washed twice with buffer containing 10 mM Tris (pH 7.6)-0.5 M NaCl-1 mM EDTA0.5% Triton X-100, twice with a similar buffer containing 0.15 M NaCl, and finally twice with detergent-free buffer. Washed beads were extracted in 50 pul of SDS buffer (62.5 mM Tris [pH 6.8]-10% glycerol-0.004% bromophenol blue2.5% 3-mercaptoethanol) at 95°C and analyzed by 5 to 15% SDS-PAGE by the method described previously (11). Gels were treated with Fluoro-Hance (Research Products International Corp.), dried, and exposed with Kodak XAR-5 film for fluorography at -70°C. Enzyme-linked immunosorbent assays (ELISAs). Immulon2 plates (Dynatech Laboratories, Inc.) were coated with 100 pul of solutions (1 jig/ml) of each peptide dissolved in 0.05 M carbonate buffer (pH 9.6) overnight at 4°C. Wells were blocked with 1% bovine serum albumin in phosphatebuffered saline-Tween 20 (0.05%) for at least 3 h before incubation (1 h) with sera. Plates were washed with phosphate-buffered saline-Tween 20 and incubated with 100 RI of diluted (1:1,000) goat anti-human immunoglobulins G, A, and M for 1 h. Washed wells were developed with ophenylenediamine as the substrate. NHS from six donors were used as the negative controls. A450 values greater than the average plus 3 standard deviations of the values obtained with NHS (cutoff line) are considered positive. All the sera were tested in triplicate. DNA isolation and Southern analysis. DNA from cultured P. falciparum parasites was purified for Southern analysis (13). DNA (2 pug) from each parasite isolate was digested with the restriction enzymes EcoRI or DraI or with mung bean nuclease in the presence of 40% formamide (14) and separated by electrophoresis on two 1% agarose gels. DNA in the gels was transferred to nitrocellulose membranes for Southern analysis with the nick-translated DNA of P. falciparum clones Pfhsp and Pfgrp (10). Hybridization was done

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FIG. 1. Detection of antibodies against Pfgrp (A) and Pfhsp (B) in human sera from Papua New Guinea by ELISA. ELISA plates were coated with the peptides and treated with sera as described in Materials and Methods. Each closed circle represents the mean of triplicate values for different sera. Bars, Mean value plus or minus the standard errors of the means of six NHS used as negative controls. The cutoff value (control plus 3 standard deviations) is represented by the dashed horizontal line.

RESULTS

at 65°C in a solution containing Sx SSC (lx SSC is 0.15 M

NaCl plus 0.015 M sodium citrate), 5 x Denhardt solution (1% Ficoll [Pharmacia Fine Chemicals]-1% polyvinyl pyrrolidine-1% bovine serum albumin), 0.1% SDS, and 100 ,ug of sonicated salmon sperm DNA per ml. Filters were washed at 500C with 0.2x SSC-0.1% SDS, wrapped in Saran Wrap, and exposed for autoradiography with Kodak XAR-5 film at -700C. Lymphocyte proliferation assay. Peripheral blood mononuclear cells (PBMC) from donors (seropositive for antibodies against various P. falciparum antigens including circumsporozoite protein and antigens in the asexual and sexual stages [7; unpublished observations]) living in Papua New Guinea were purified from heparinized blood by centrifugation through Ficoll-Hypaque, stored, and shipped (in RPMI 1640 medium containing 10% fetal calf serum and 7.5% dimethyl sulfoxide) frozen in liquid nitrogen. PBMC were quickly thawed at 370C and washed once in the culture medium; the viability of thawed PBMC in various experiments was between 90 and 95%. PBMC from normal healthy volunteers in the laboratory were similarly purified for proliferation assays. Cells (2 x 105 in 100 ,ul per well of round-bottomed, 96-well NUNC plates) were cultured in triplicate in RPMI 1640 medium containing 25 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), penicillin, streptomycin, and 10% heat-inactivated (30 min, 56°C) AB (Rh') serum in a CO2 incubator at 37°C. Cells were pulsed with 1 ,uCi of [3H]thymidine for 16 h before being harvested either on day 3 for mitogens or on day 7 for antigens. Cells were harvested on a glass fiber sheet with a Skatron cell harvester. Radioactivity in the filter disks was determined by liquid scintillation counting. Results are expressed as the stimulation index (SI) and calculated as follows: SI = (counts per minute in the presence of antigen background counts per minute)/(counts per minute without antigen background counts per minute). -

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Detection of antibodies against parasite stress proteins in the from people exposed primarily to P. fakciparum. Synthetic peptides based on unique sequences in the parasite stress proteins, the GGMP repeat sequence in Pfhsp, and the carboxy-terminal sequence in Pfgrp were used in an ELISA to detect specific antibodies. Titration of the peptide concentration with rabbit anti-Pfhsp or anti-Pfgrp sera suggested 1 ,ug/ml as the optimum concentration for coating the wells of the ELISA plates. Human sera from Papua New Guinea at 1:100 and 1:200 dilutions were tested in triplicate wells. Each plate also contained equivalent dilutions of six different NHS as negative controls. The control sera were obtained from people who have never been to any malaria-endemic areas. Of 21 sera, 8 (38%) contained antibodies reacting with the single peptide of the Pfgrp sequence (Fig. 1A). On the other hand, 62% of these sera (13 of 21) contained antibodies recognizing the repeat sequence present in Pfhsp (Fig. 1B). The data shown in Fig. 1 do not include other epitopes present in these proteins and likely to be immunogenic. Of the total sera which tested positive for either Pfhsp or Pfgrp, three-fourths was from people not infected with parasites (blood smear negative) at the time of collection. Although the total number of subjects in various age groups is small, no obvious age-related trend was apparent. Table 1 shows the average ELISA values for the three age-group breakdowns of the total number of samples. To further confirm the presence of antibodies against stress proteins, the sera were employed in immunoprecipitation analysis with a detergent extract of metabolically labeled parasite. All the human sera recognized a large number of parasite antigens including a prominent protein band in the region of 70 to 75 kDa (Fig. 2A, arrow). Sera that gave higher ELISA readings also showed more intense 75-kDa bands (Pfhsp). Proteins with similar molecular sera

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organization of genes for Pfhsp- and Pfgrp-like proteins comes from the data shown in Fig. 3. DNA from cultured parasites 3D7 (a clone of NF54 isolated from a worker living near the Amsterdam airport), 7G8 (a clone of Brazilian isolate IMTM22), HB3 (a clone of an isolate from Honduras), ItG2F6 (a clone of another Brazilian isolate, Ituxi), Ni (an isolate from Nigeria), LE5 (a clone derived from a Liberian isolate), and T2 (a clone derived from a Thai isolate) was digested with restriction enzymes or mung bean nuclease, separated on agarose gels, transferred onto nitrocellulose membranes, and probed with Pfhsp- or Pfgrpspecific labeled DNA. The pattern of hybridization to major bands was not different among various DNA samples (Fig. 3). The hybridization bands seen with mung bean nucleasedigested DNA showed slight variations in size. This could probably be the result of differences in the sites in DNA from various isolates cut by this enzyme. These genes are therefore conserved in parasites from different geographical areas. In addition, the immunoprecipitation of biosynthetically labeled parasite extracts showed proteins corresponding to stress proteins (Fig. 2 [for 3D7]) (10 [for 7G8]) (data not shown [for other parasites]). Sera from people exposed to P. vivax do not react with Pfhsp and Pfgrp peptides. Sera from people who were living in Guatemala and had been exposed to P. vivax were similarly tested by ELISA with the Pfhsp and Pfgrp peptides. These sera have been shown to cross-react with many antigens found in the blood-stage P. falciparum parasites but not with the repeat sequence of the P. falciparum circumsporozoite protein (J. P. Folgar, P. Lubega, and N. Kumar, unpublished observations). None of the sera reacted with the Pfgrp

TABLE 1. Age distribution of sera which are Pfhsp and Pfgrp peptide positive A450 (mean +

Age group

(yr)

Pfhsp

10-20 20-40 40-60

0.44 + 0.016 (2) 0.35 ± 0.016 (5) 0.47 ± 0.112 (4)

a

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Pfgrp

0.542 + 0.29 (3) 0.51 ± 0.104 (3) 0.551 ± 0.162 (2)

Numbers in parentheses represent the total number of samples.

masses were immunoprecipitated by rabbit antisera against Pfhsp or Pfgrp fusion proteins or Pfgrp peptide conjugated to keyhole limpet hemocyanin. The data in Fig. 2B confirm the identity of the 70- to 75-kDa proteins immunoprecipitated by the human sera as the stress proteins and not any other proteins with similar electrophoretic mobilities. Extracts were first immunodepleted four times with the rabbit prebleed (Fig. 2B, lanes 1 and 2) or rabbit antisera against the Pfhsp fusion protein (Fig. 2B, lanes 5 to 8). Immunodepleted extracts were then tested with two of the human sera (A19 and A54). A comparison of immunoprecipitation by human sera after depletion with normal rabbit serum (lanes 1 and 2) and rabbit anti-Pfhsp serum (lanes 9 and 10) clearly establishes that the 75-kDa band seen in the immunoprecipitations with human sera is Pfhsp. Genes for Pfhsp and Pfgrp are conserved in parasites from various geographical locations. Sequences of Pfhsp and Pfgrp reported in four different isolates of P. falciparum either are the same or contain minor replacements (2, 3, 10, 27). Further evidence for the presence and similar genomic

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