Proc. Natl. Acad. Sci. USAVol. 80, pp. 3787-3791, June 1983Immunology

Expression of Plasmodium falciparum blood-stage antigens inEscherichia coli: Detection with antibodies from'immune humans

(malaria/recombinant DNA/bacteriophage A/j&galactosidase/fused polypeptides)

DAVID J. KEMP, Ross L. COPPEL, ALAN F. COWMAN, ROBERT B. SAINT, GRAHAM V. BROWN, ANDROBIN F. ANDERSThe Walter and Eliza Hall Institute of Medical Research, Post Office, The Royal Melbourne Hospital, Victoria 3050, Australia

Communicated by G. J. V. Nossal, March 23, 1983

ABSTRACT Many proteins produced by blood stages of themalaria parasite PlAsmnodiumfalciparum are natural immunogensin man. As an approach to determining which of these are rele-vant to protective immunity we have constructed an expressionlibrary of P. falciparum cDNA sequences, cloned in Escherichiacoli The cDNA sequences were inserted into the fi-galactosidasegene of an ampicillin-resistant derivative of the temperature-sen-sitive lysogenic bacteriophage Agtll. About 5% of the resultingclones expressed P. falciparum sequences as polypeptides fused to(8-galactosidase. We have identified many clones that express P.falciparum antigens by immunological screening in situ with an-tibodies from immune human sera that inhibit P. falciparum growthin vitra The antigen-positive clones contain P. falciparum cDNAsequences, as determined by hybridization. Some express poly-peptides that are larger than 3-galactosidase and react both withantibodies to (3-galactosidase and with antibodies from humansimmune to P. falciparum. The cloned P. falciparum antigens shouldfacilitate new approaches to the identification of potential vaccinemolecules.

An effective vaccine against Plasmodiumfalciparum, the caus-ative agent of the lethal form of human malaria, will almost cer-tainly include protective antigens from both the sporozoite andblood stages of the parasite (1, 2). Sporozoites, the infectivestage from the mosquito, elicit a response to only one dominanthost-protective antigen (2). In contrast, the blood stages of P.falciparum contain a plethora of natural immunogens, so thatsera from adults who have acquired antibody-mediated im-munity by continuous exposure react with many blood-stageparasite proteins (3). The number of these antigens that are pro-tective remains to be determined, although some candidatemolecules have been identified (4-6). Further, the purificationof such antigens by conventional procedures from parasites grownin vitro (7) is seriously limited by the requirements for humanerythrocytes and serum in the culture system.We have attempted to circumvent these problems by using

recombinant DNA techniques to express a wide range of an-tigens from blood stages of P. falciparum in Escherichia coli.We describe here the construction of a library of cloned P. fal-ciparum cDNA sequences that are expressed in E. coli as poly-peptides fused to B3-galactosidase (,&Galase) and the identifi-cation of many clones that express P.falciparum antigens. Theseclones, which represent "monoclonal antigens," will facilitatenew experimental approaches to the analysis of immunity to P.falciparum.

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

MATERIALS AND METHODSReagents. DNA polymerase I, EcoRI, and Xba I were from

Boehringer Mannheim. EcoRI methylase, BstNI, exonucleasesBAL-31 and S1, calf intestinal phosphatase, and T4 ligase werefrom New England BioLabs. Reverse transcriptase was the giftof J. Beard (Life Sciences, St. Petersburg, FL). EcoRI linkers(C-G-G-A-A-T-T-C-C-G) were from New England BioLabs, XbaI linkers (C-T-C-T-A-G-A-G) were from Amersham (England).,-Galase (chromatographically purified) was from P-L Bio-chemicals.

Strains. Bacteriophages Agtl1 and Agt10 and E. coli strainsRY1073 and RY1082 (8) were the generous gifts of R. Young,T. Huynh, and R. Davis (Stanford University).

P. falciparum mRNA. Isolate FCQ-27/PNG of P. falcipa-rum from the Madang area of Papua New Guinea was estab-lished in this laboratory in 1979 and maintained in continuousculture (7). Enriched parasite preparations were prepared bylysis of the erythrocytes with saponin (9). RNA was extractedfrom the parasites by homogenization in 10 vol of 6 M guanidin-ium-HC/0. 1 M NaOAc, pH 5.2, and centrifugation through4.8 M CsCl in 10 mM EDTA (pH 8.0) at 35,000 rpm overnightin a Beckman SW 40 rotor. Poly(A)+ RNA was selected byoligo(dT)-cellulose chromatography (10).

Construction of cDNA Clones in AgtlO. Standard proce-dures were followed for enzymic reactions (11). mRNA (-4 jig)was copied into cDNA with reverse transcriptase and double-stranded cDNA was prepared with DNA polymerase I. Aftertreatment with nuclease S1, the cDNA was methylated withEcoRI methylase and ligated to 0.5 ,ug of phosphorylated EcoRIlinkers with T4 ligase. The mixture was then cleaved with EcoRIand fractionated on a 1% agarose gel in 50 mM Tris/20 mMNaOAc/2 mM EDTA, pH 8.2. DNA between 0.6.and 2.0 kilo-bases (kb) was recovered by electrophoresis onto a DEAEmembrane filter (Schleicher & Schuell, NA45), eluted in 1 MNaCI/50 mM arginine (free base) at 70°C for 1 hr, and precip-itated with ethanol. Aliquots (20 ng) were ligated to EcoRI-cleaved AgtlO DNA (1 ,g), packaged into phage, and plated onE. coli RY1073 (8). About 2 X 105 recombinants were obtained,and "'40% of these hybridized detectably to [32P]cDNA fromP. falciparum.

Construction of Agtll-Amp3 (AAmp3). DNA (5 /ig) frompBR322 was cleaved with EcoRI and BstNI. After treatmentwith 0.3 unit of exonuclease BAL-31 for 30 sec to remove pro-truding ends, the DNA was ligated to Xba I linkers as above,cleaved with 100 units of Xba I, and fractionated on a 1% agar-

Abbreviations: ,B-Galase, ,B-galactosidase; AAmp3, Agtll-Amp3; kDa,kilodalton(s); kb, kilobase(s); AmpR, ampicillin-resistant.3787

Proc. Nati. Acad. Sci. USA 80 (1983)

ose gel. The 1.7-kb fragment was recovered and ligated to 1,ug of Xba I-cleaved Agtll DNA (8) and packaged as above. E.coli RY1082 was infected with the phage and plated at 30°C onL plates containing ampicillin (30 ,ug/ml). An ampicillin-re-sistant (AmpR) colony was chosen and shown to be lysogenic fora lac+, temperature-sensitive phage designated AAmp3, whichis identical to Agtll except for an =1.7-kb fragment from pBR322inserted at the Xba I site.

Insertion of Amplified cDNA From AgtlO into AAmp3. DNA(500 ug) prepared from plate stocks of the AgtlO-cDNA librarygrown on E. coli C600 r k, m k was cleaved with EcoRI (1,000units) for 2 hr at 37°C and 1/50th of the total was labeled with[32P]dATP and the Klenow fragment of DNA polymerase I. Afterphenol extraction, the labeled and unlabeled DNAs were mixedand centrifuged (18 hr, 37,000 rpm in a Beckman SW 40 rotor)on a 10-40% glycerol gradient in 10 mM Tris HCI, pH 7.4/1mM EDTA/300 mM NaOAc. The cDNA was detected as a ra-dioactive peak and recovered by ethanol precipitation.

Uncut AAmp3 DNA (50 ,g in 100 ,ul) was ligated for 4 hr at150C to protect the cohesive ends from subsequent phospha-tase treatment. The ligase was inactivated at 70°C for 10 min,NaCl and Tris HCI at pH 7.4 were added to 50 and 100 mM,respectively, and the DNA was cleaved with EcoRI, treated withcalf intestinal phosphatase (0.02 unit/,ug, 30 min), extractedwith phenol, and precipitated with ethanol. Aliquots (1 ,ug, 100pg/ml) were ligated for 16 hr at 150C to 80 ng of amplifiedcDNA and packaged as above. E. coli RY1082 was infected withthe packaged phage for 30 min at 30°C, incubated at 30°C fora further 30 min after addition of L broth (1 ml) to allow expres-sion of ,B-lactamase, and plated on nitrocellulose filters on Lplates containing 30 ug of ampicillin per ml at 30°C. AAmp3-cDNA lysogens can be directly selected from RY1082 cells in-fected with the packaged phage because the cDNA has been

modified by growth on E. coli C600 rj, mj (RY1082 is rk).Screening of the Amp3-P. falciparum cDNA Library with

Antibodies to P. falciparum. Colonies were replicated to nitro-cellulose filters, grown for 1-2 hr at 30'C on CY plates (or, inmore recent experiments, L plates) containing 30 ;kg of am-picillin per ml, and induced at 420C for 1.5 hr. The colonieswere lysed by placing the filters on 3MM paper saturated with1% NaDodSO4 in H20 for 15 min and then for 15 min in anatmosphere saturated with CHCl3. The filters were washed byrocking in 10 mM Tris HCI, pH 8.0/0.15 M NaCl (Tris/NaCI)containing 3% bovine serum albumin for 2 hr and then in Tris/NaCl alone for 1 hr at room temperature. They were then treatedwith affinity-purified anti-plasmodium antibodies (25 ug/ml)in bovine serum albumin/Tris/NaCl for 2 hr, washed once inTris/NaCl, twice in Tris/NaCl containing 0.05% Triton X-100,and again in Tris/NaCl, treated with "2I-labeled protein A fromStaphylococcus aureus (specific activity = 40 ,uCi/,g; 0.5 ,uCi/ml; 1 Ci = 3.7 x 1010 Bq) in bovine serum albumin/Tris/NaClfor 1 hr, washed, dried, and autoradiographed with an inten-sifying screen for 16 hr.

Preparation and Purification of Sera. Serum was collectedwith informed consent from adults free of P. falciparum in theendemic Madang area of Papua New Guinea. IgG isolated onprotein A-Sepharose from each serum was tested for inhibitionof growth of P. falciparum in vitro (4) and five inhibitory serawere pooled. The antibodies were purified by two cycles ofabsorption to P. falciparum (isolate FCQ-27/PNG) proteinsbound to CNBr-activated Sepharose. For this, a parasite con-centrate prepared by saponin lysis was sonicated in phosphate-buffered saline and centrifuged at 2,000 x g for 10 min. Thesupernatant was adjusted to =20 mg/ml with phosphate-buff-ered saline and conjugated to CNBr-Sepharose. Bound anti-bodies were eluted with 0.1 M glycine/0. 15 M NaCl, pH 2.6,

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FIG. 1. Construction of a P. faiciparum cDNA expression library (schematic diagram). Insertion of the ,B-lactamase gene (which codes for am-picillin resistance) frompBR322 into Agtll, generating AAmp3, is depicted (top right). Synthesis ofdouble-stranded (ds)P. falciparumcDNAbearingEcoRI linkers is shown on an -5-fold greater scale (center). This cDNAwas cloned in AgtlO to amplify and methylate it in the pattern characteristicofE. coli K-12 (left). Amplified cDNA isolated from the AgtlO-cDNA library was inserted into EcoRI-cleaved, dephosphorylated AAmp3 DNA (bot-tom). Lysogens were generated by infecting E. coli RY1082 and plating for AmpR colonies. A fused polypeptide bearing most of 3-Galase as its NH2terminus and a P. falciparum polypeptide as its COOH terminus is shown (bottom).

Lac'AmpR

3788 Immunology: Kemp et al.

Proc. Natl. Acad. Sci. USA 80 (1983) 3789

A B

I

C D

FIG. 2. Detection ofP. falciparum antigens expressed inE. coli. (A)About 103 AAmp3-cDNA colonies were lysed in situ and screened withimmune human IgG. (B)A replica ofthe coloniesshown inA was screenedwith nonimmune human IgG (obtained from individuals in Melbourne,Australia, with no history of possible exposure to P. falkiparum). (C)Positive colonies detected as inA (generally contaminated by 3-10 sur-rounding negative colonies) were streaked for single colonies; for eachisolate, 12 single colonies picked at random were grown in triplicateand rescored as in A. All positive colonies in each successive set of tworows derive from one original colony. (D)A replica ofthe colonies shownin C was screened with nonimmune IgG as in B.

and immediately adjusted to pH 7.4 with 2 M Tris HCl. Thepurified antibodies immunoprecipitated a spectrum of 'S-la-beled P. falciparum proteins similar to that precipitated by theserum.

Analysis of Proteins by Electrophoretic Transfer Blotting.Protein extracts of induced AAmp3-P. falciparum clones wereprepared and fractionated on 7.5% polyacrylamide/NaDodSO4gels. Proteins from the gels were transferred electrophoreti-cally to nitrocellulose (12) and incubated for 1 hr at room tem-perature in bovine serum albumin/Tris/NaCl before reactionfor 90 min with rabbit anti-(3-Galase or anti-P. falciparum an-tibodies. Antibodies to E. coli in the human IgG were removedby incubating 0.5-1.0 mg of human IgG with 1 ml of sonicateof AAmp3-infected E. coli RY1082 for 1 hr at 40C. The super-natant obtained by centrifugation (12,000 X g, 10 min) was di-luted with bovine serum albumin/Tris/NaCl to 50 ml. The ni-trocellulose sheets were then washed, reacted with 'WI-labeledprotein A, rewashed, and autoradiographed as above. Rabbitantiserum to /3-Galase was prepared by injection of 0.5 mg of,(3Galase in complete Freund adjuvant (subcutaneously and in-tramuscularly), followed at 4-wk intervals by injection of 0.5 mgin incomplete Freund adjuvant. Serum collected 2 wk after thethird injection was used after absorbing out antibodies reactiveto E. coli RY1082 as above.

By using high-frequency-lysogeny E. coli strains (8), most col-onies obtained after infection with phage are lysogens. How-ever, residual nonlysogenic colonies, being lac, are difficultto distinguish from recombinant lysogens. To overcome thisproblem we introduced the ,(3lactamase gene from pBR322 intoAgtll (see Fig. 1 and Materials and Methods), generating a phagedesignated AAmp3. Lysogens of AAmp3-cDNA clones giveAmpRlac- colonies, readily distinguishable from parentalAmpRlac+ colonies on plates containing ampicillin and a ,(3Gal-ase indicator (5-bromo-4-chloro-3-indolyl-,( D-galactopyrano-side), whereas nonlysogens are killed. To prevent religated pa-rental molecules predominating in our library (8), the EcoRI-cleaved AAmp3 vector DNA was treated with phosphatase. Be-cause this markedly decreased the cloning efficiency, we firstamplified the cDNA by cloning it into the phage vector AgtlO(Fig. 1).

In the resulting library of -5 X 104 lysogenic AmpR colo-nies, >98% were lack and 70% hybridized detectably to [32P]_cDNA from P.falciparum. A survey of >100 clones by gel elec-trophoresis revealed that U5% produced fused polypeptidesmore than 5 kilodaltons (Dad longer than (3-Galase (not shown).

Identification of E. coli Colonies that Express P. falciparumAntigens. To detect antigen-positive colonies we lysed inducedcolonies growing on nitrocellulose filters under conditions thatallowed binding of -proteins directly to the filters. After re-maining binding sites had been blocked with bovine serum al-bumin, the filters were treated with antibodies, followed bytreatment with "5I-labeled protein A from S. aureus. Similarprocedures have been described elsewhere (8, 13, 14). The an-tibodies were isolated from the sera of putatively immune PapuaNew Guineans. IgG isolated from these sera on protein A-Sepharose inhibited the growth of P. falciparum in vitro. Be-cause this IgG also reacted strongly with E. coli, antibodies werepurified by affinity chromatography on P. falciparum proteins,followed by depletion of the anti-E. coli activity (see Materialsand Methods).

The result of such a screen on a filter containing about 103AAmp3-cDNA colonies is shown in Fig. 2 A and B. At least 10colonies reacted detectably with immune IgG but not with nor-

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RESULTS AND DISCUSSIONOur strategy for construction of an expression library from P.falciparum mRNA, based on the Agtll expression system ofYoung and Davis (8), is shown in Fig. 1. This system allows cDNAcloned into the unique EcoRI site near the 3' end of the /3-Gal-ase gene of Agtll to be expressed as a polypeptide fused to ,-Galase. E. coli colonies lysogenic for the temperature-sensitiverecombinant phage can be grown at the permissive tempera-ture and induced at 420C to achieve high levels of expression.

FIG. 3. Hybridization of P. falciparum cDNA to antigen-positivecolonies.Areplicaofthe coloniesshown in Fig. 2Cwasscoredwith [32P]-cDNA (3 x 106 cpm/ml) prepared from P. falciparum mRNA with re-verse transcriptase, oligo(dT), and [32P]dCTP (specific activity = 3,000Ci/hmol). The corresponding areas of filters scored with immune IgG(anti-P. falciparum, A) and with cDNA (B) are shown.

Immunology: Kemp et al.

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Proc. Natl. Acad. Sci. USA 80 (1983)

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FIG. 4. Detection of P. fakciparum-3-Galase antigenic fused poly-peptides. Extracts from RY1082 lysogenic for the AAmp3 vector (V) forantigen-positive clones Ag8, Agl3, Agl5, Ag21, and Ag23 and for con-trol (antigen-negative) clones C5, C9, and C13 were prepared from in-duced 1-ml cultures by boiling for 2 min in sample buffer (8) and frac-tionated on 7.5% NaDodSO4 gels. (A) Gel stained with Coomassie blue.(B) The proteins were transferred to nitrocellulose (12) and probed withanti-(-Galase serum. (C) As for B, but probed with the same immunehuman IgG as in Fig. 2. Autoradiography was for 3 hr (B) and 60 hr (C).The sizes of some (-Galase polypeptides are shown in kDa.

mal IgG. The range of signal intensities with immune IgG var-ied widely and both immune and normal IgG gave backgroundreactions, which limit detection of weakly positive colonies. Afterpurification by plating, many of the colonies again reacted withthe immune IgG (Fig. 2C) but not with normal.IgG (Fig. 2D).The signals were reproducible in the triplicates and the inten-sity of signals in any row (all of which derive from the' sameoriginal positive colony) was characteristic. All of the antigen-positive colonies were lac-.

The Antigen-Positive Colonies Contain P. falciparum cDNASequences. When tested by colony hybridization with P. fal-ciparum [rP]cDNA sequences, all of the antigen-positive cloneshybridized (Fig. 3). The extent of hybridization varied mark-edly with different clones, as would be expected if the clonesoriginate from different mRNAs. In general, the antigen-pos-itive clones hybridized weakly and hence are derived from

mRNAs of moderate abundance. There was no relationship be-tween the extents of immunological reactivity and hybridiza-tion (Fig. 3). We conclude that most positive clones derive fromindependent cloning events.

P. falciparum Antigens Expressed as Polypeptides Fused tofB-Galase. We examined proteins from lysates of >50 inducedantigen-positive clones by polyacrylamide gel electrophoresis.Staining with Coomassie blue revealed that -20% of them pro-duced abundant unique polypeptides larger than (3-Galase (e.g.,antigen-positive clones Ag13 and Ag23 in Fig. 4A). Hence, thecolony assay has selected for clones producing large fused poly-peptides. When the proteins were transferred from gels to ni-trocellulose and probed with anti-(3-Galase serum, the 3-Gal-ase marker from the parental lysogen RY1082-AAmp3 wasdetected as a major band of 116 kDa (Fig. 4B). The smallerfragments in this gel are degradation products of /3-Galase, asnone was present in the' lac- strain RY1082. The unique largepolypeptides from clones Ag13 and Ag23 and from control cloneC9 all reacted strongly with anti-f3-Galase serum (Fig. 4B). P-Galase degradation products were produced by all of the clones,but no other polypeptides significantly larger than (Galase weredetected (Fig. 4B). Clone Ag23 contained ,B-Galase polypep-tides of 142, 127, and 118 kDa (Fig. 4B), which presumablyarise by intracellular proteolysis of a 160-kDa precursor (seebelow). Several clones contained low levels of fused polypep-tides and high levels of 116- to 118-kDa polypeptides (not shown).Hence, it is probable that in clones such as Ag8, Agl5, and Ag2l,the fused polypeptides were degraded. Alternatively, somepositive clones may contain polypeptides that result from in-ternal initiation (13, 15, 16).When equivalent filters were probed with the affinity-pu-

rified anti-P. falciparum IgG, three different results were ob-tained (Fig. 4C). First, the 150-kDa polypeptide from cloneAgl3 reacted strongly, demonstrating that it contains both a P.falciparum antigenic determinant and ,B-Galase sequences. Mi-nor amounts of a 162-kDa polypeptide were also detected (Fig.4C). As expected, no reaction was observed with the 122-kDa,fGalase polypeptide in control clone C9 (Fig. 4C). Second,antigen-positive clone Ag23 gave a positive reaction but therewas a smear of reactive material, ranging up to 160 kDa (Fig.4C). We presume that the 160-kDa polypeptide is highly un-stable, even though E. coli RY1082 bears the lon- mutation,which decreases proteolysis (8). The abundant 127-kDa and 142-kDa ,B-Galase polypeptides of Ag23 (Fig. 4B) probably rep-resent relatively stable degradation products that lack the P.falciparum antigenic determinant. Third, in the remaining clonesno polypeptides reacted demonstrably with immune human IgG(Fig. 4C). This may reflect degradation or loss of antigenicityafter boiling in NaDodSO4/2-mercaptoethanol.

CONCLUSIONSWith P. falciparum, purification of individual proteins is ham-pered by the small amounts of material available. Further-more, it is not clear which of the many immunogens are im-portant in naturally acquired host-protective immunity. Hence,we have used antibody preparations from humans containingmany anti-P.falciparum specificities to isolate cDNA clones ex-pressing different P.falciparum antigens. We estimate that ='1%of the clones in our library are antigen-positive and thus thelibrary contains several hundred positive clones, Because thecDNA was amplified, not all will represent independent clon-ing events. However, the data in Figs. 3 and 4 suggest that mostof the clones examined so far are distinct and originate from avariety of mRNAs. Hence, the antigen-positive clones in our

3790 Immunology: Kemp et al.

w -N

Proc. Natd. Acad. Sci. USA 80 (1983) 3791

library may represent a significant fraction of the immunogenicproteins of P. falciparum.

Each of the colonies that we have detected represents a"monoclonal antigen" of P.falciparum. The battery of such an-tigens provided by our library will facilitate the analysis of im-munity to P. falciparum in several ways. For example, the re-actions of sera from a spectrum of individuals of differingimmunological status with the battery of cloned antigens mayreveal those antigens relevant to protective immunity. More-over, it should be feasible to prepare antisera to the fused poly-peptides and to test the sera for reactivities such as surfacebinding, inhibition of invasion, or growth of P. falciparum invitro. Polyclonal antibodies prepared against a monoclonal an-tigen should differ significantly from a monoclonal antibody be-cause, in many instances, they should react with more than oneepitope. This property may aid in the identification of antigenswith protective value. Similar approaches to the identificationof relevant antigens should be possible with other parasite sys-tems.

We thank R. Young, T. Huynh, and R. Davis for generous gifts ofstrains and communication of results before publication, G. F. Mitch-ell, E. Handman, and T. Spithill for helpful discussions, L. Corcoranfor the packaging mix, and A. Edwards, K. Easton, L. Gibson, J. Thomas,and M. Horsfield for expert technical assistance. We thank M. Alpers,P. Heywood, and other members of the Papua New Guinea Instituteof Medical Research for collaboration in obtaining serum and parasitesamples from Papua New Guinea. This work was supported by the Aus-tralian National Health and Medical Research Council, the Rockefeller

Foundation Great Neglected Diseases Network, and the United Na-tions Development Program/World Health Organization/World BankSpecial Program for Research and Training in Tropical Diseases.

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Immunology: Kemp et al.