Vol. 59, No. 10INFECTION AND IMMUNITY, OCt. 1991, p. 3498-35030019-9567/91/103498-06$02.00/0Copyright © 1991, American Society for Microbiology

Ability of Recombinant or Native Proteins To Protect Monkeysagainst Heterologous Challenge with Plasmodium falciparumHOWARD M. ETLINGER,* PATRICK CASPERS, HUGUES MATILE, HANS-JOACHIM SCHOENFELD,

DIETRICH STUEBER, AND BELA TAKACSCentral Research Units, F. Hoffmann LaRoche Ltd., CH4002 Basel, Switzerland

Received 18 December 1990/Accepted 10 July 1991

To circumvent problems associated with polymorphic vaccine candidates for Plasmodium falciparummalaria, we evaluated recombinant proteins representing sequences from relatively highly conserved regions ofthe precursor to the major merozoite surface proteins, gp190, for their ability to protect Saimiri monkeysagainst malaria challenge. Recombinant proteins represented amino acid residues 147 to 321 (p190-1) or 147to 321 and 1060 to 1195 (p190-3), and their efficacy was compared with that of native gpl90 and its processedproducts. All antigens were derived from P. falciparum Ki, a Thai isolate, while the challenge strain was PaloAlto (from Uganda, Africa), which contains, with the exception of the N-terminal 375 amino acids, which arealmost identical to the Kl sequence, essentially the MAD-20 allelic form of gpl90. By 12 days followingchallenge, each control monkey required drug treatment. Three monkeys injected with p190-3 requiredtherapy, while one cleared the parasites without therapy. Two monkeys injected with p190-1 received therapyon day 14, while the remaining two cleared the parasites without therapy. Of four animals injected with nativegpl90, because of health reasons unrelated to malaria, one was not challenged with parasites and one wasremoved from the study 8 days after challenge when its parasitemia was 1.1% (parasitemias in control animalsranged from 4.3 to 9%); the remaining two cleared the parasites after maximum parasitemias of 0.45 and0.53%. The highest levels of antiparasite antibody were produced by animals immunized with native gpl90.There was a significant correlation between monkeys which did not require drug treatment and antiparasiteantibody. These results may suggest that native gpl90 and/or its processed products can provide excellentprotection against heterologous challenge and that antibody is important for protection. The challenge forvaccine development is to identify the protective sequence(s).

One of the difficulties associated with the development ofa successful malaria vaccine is the presence of parasitevariability, which has been postulated to inhibit the appear-ance of effective immunity (1). For example, in the case ofone group of vaccine candidates against the sporozoite stageof the human malaria parasite, Plasmodium falciparum, thecircumsporozoite proteins, the immunodominant sites forhuman T cell recognition, are polymorphic (11). A secondgroup of vaccine candidates also characterized by polymor-phism are the major merozoite surface proteins of P. falci-parum. These are derived from a precursor protein of about190 kDa, and the variability of gp190 reflects, in part, thepresence of two prototypic forms of the gene, found inisolates Kl and MAD-20 (3, 6, 13, 15, 16, 18-21, 24-26, 28,30, 35, 36). In addition to dimorphic segments, the proteincontains relatively highly conserved as well as polymorphicregions. Purified gp190-derived proteins have been shown insome instances to eliminate the need for drug therapy inimmunized monkeys challenged with P. falciparum (12, 29,32). The monkey protection experiments which have beenperformed to date have utilized both heterologous andhomologous parasite challenges. Since some areas of gp190are relatively highly conserved, the term heterologous isused in the present study to indicate that the gp190-derivedsequences used for immunization and those carried by thechallenge parasite are not identical. The best protection wasprovided by gp190 and its processed products (native gpl90)when monkeys were challenged with parasites carrying thesame gpl90 as that used for immunization (32). Since an

* Corresponding author.

ideal vaccine will provide a broad degree of protection, wehave evaluated the ability of recombinant proteins contain-ing sequences from relatively highly conserved areas ofisolates Ki and MAD-20 to protect against heterologouschallenge. The efficacy of native gp190 to protect againstheterologous challenge was also determined.

MATERIALS AND METHODS

Immunogens. (i) Recombinant proteins. DNAs coding forp190-1 (amino acid residues 147 to 321) and p190-3 (aminoacid residues 147 to 321 and 1060 to 1195), representinghighly conserved regions of the Kl isolate, were cloned andexpressed in Escherichia coli as previously described (9).p190-1 is similar to 190 L (33) and each recombinant proteindescribed in this report had an additional 4 to 6 amino acidsresulting from the cloning. p190-1 contained 6 histidine(hexa-His) residues at the amino end, while p190-3 had ahexa-His sequence at the carboxy end. The recombinantproteins were purified to homogeneity from E. coli lysates bya combination of chromatofocusing and Ni2'-chelate affinitychromatography as described previously (33) or by succes-sive hydrophobic interaction chromatography, Ni2+-chelatechromatography, ion-exchange chromatography, and gel fil-tration chromatography. Each recombinant protein wasjudged to be at least 95% pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

(ii) Native gp190. Native gpl90 was purified by immunoaf-finity chromatography from a lysate of P. falciparum Kicultured in human A' erythrocytes as described previously(27). Nonsynchronous cultures were harvested when para-sitemia reached 10 to 15%, and parasites were isolated after

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saponin lysis of erythrocytes (10). Parasite pellets wereextracted with 10 volumes of 1% Nonidet P-40 in 5 mMTris-Cl (pH 7.2)-50 mM NaCl-5 mM EDTA-5 mM ethyleneglycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid-1mM phenylmethylsulfonyl fluoride-100 U of aprotinin(Trasylol) per ml-10 ,ug of leupeptin per ml for 60 min on ice.Lysates were centrifuged at 48,000 x g for 30 min, and thesupernatant fractions were passed through a column contain-ing 2 ml of Sepharose 4B coupled to protein A-purified rabbitimmunoglobulin. This immunoglobulin was from an animalinjected with a Ki-derived recombinant protein containingp190-3 amino acid residues 1060 to 1195 and 147 to 321 (190N) (33). To remove nonspecifically bound proteins, wewashed the column with 50 volumes of 50 mM Tris-Cl (pH7.4) containing 0.3 M NaCl, 0.5% Nonidet P-40, and 100 U ofaprotinin per ml. Bound proteins were eluted with 0.2 Mglycine-HCl (pH 2.8) containing 0.5% Nonidet P-40 and 0.3M NaCl. Fractions containing gp190, as determined by dotblotting, were pooled and concentrated in collodion bags(Sartorius) with N2 pressure.

Immunization. Four Saimiri monkeys each received sub-cutaneously 100 ,ug of p190-1 or p190-3 in Freund's completeadjuvant on day 1 and 100 jig of the same antigen in Freund'sincomplete adjuvant on days 24 and 48. Monkeys in a thirdgroup received approximately 8 ,ug of native gpl90 in Fre-und's complete adjuvant on day 1 and the same amount ofnative gpi90 in Freund's incomplete adjuvant on days 24 and48. Control animals received 0.9% NaCl in the adjuvants onthe same days.

Parasite challenge. On day 60, each monkey receivedintraveneously 3.5 x 107 P. falciparum (Palo Alto)-infectederythrocytes from a donor monkey. Serological typing re-sults, obtained with blood smears containing erythrocytesinfected with our parasite challenge strain and a panel of 18monoclonal antibodies, were consistent with the gpl90 se-quence of Palo Alto published by Chang et al., who foundthat, except for a 36-bp deletion in the repeat region, theN-terminal 375 amino acids of the Kl and Palo Alto proteinsare 99.9% identical; the remainder of the Palo Alto protein isvery similar to the MAD-20 protein (4). There was noevidence for parasite heterogeneity on the basis of reactivityof the monoclonal antibodies, since each monoclonal anti-body stained either all or none of the parasites. Followingchallenge, blood smears obtained daily were stained withGiemsa stain and the percentage of parasitemia was deter-mined. Animals which had 20% parasitemia received drugtherapy consisting of quinine and pyrimethamine-sulfadox-ine (Fansidar). The experiment was stopped 50 days afterparasite challenge, at which time all remaining monkeysreceived the antimalarial drugs.

Antibody analyses. Sera or plasma were collected prior toimmunizations and parasite challenge. Reactivity was eval-uated by immunofluorescence (27), enzyme-linked immuno-sorbent assay (ELISA) (5), and immunoblotting (34) andwith a Bio-Rad Mini-Protean II dual-slab-cell unit (used asdescribed by the manufacturer) with the gel and buffersystem described by Laemmli (23).Other p190 recombinant proteins. DNAs coding for recom-

binant proteins from the MAD-20 gpl90 gene were cloned,expressed, and purified from E. coli cell lysates (9). Theseproteins contained amino acid residues 123 to 302, 384 to595, 595 to 897, 1078 to 1251, 1250 to 1398, and 1397 to 1563;each construct also contained hexa-His residues on eitherthe amino- or the carboxy-terminal end. Four additionalrecombinant proteins contained amino acid residues 34 to595, 1250 to 1563, 1250 to 1679, and 1397 to 1679; they also

200

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a b c d eFIG. 1. Immunoblot analysis of native gpl90. Total P. falci-

parum detergent extracts (lanes b, d, and e) and immunoaffinity-purified gpl90 (lane c) were probed with either endemic-area plasma(lanes b and c), monkey immunoaffinity-purified gpl90 antiserumobtained prior to challenge with parasites (lane d), or rabbit 190 Nantiserum (lane e). Lane a contains '4C-labeled molecular weightstandards. Polypeptides marked with an asterisk in lanes c and drepresent immunoglobulin heavy and light chains that were elutedfrom the immunoaffinity column and elicited the production ofantibodies.

had hexa-His residues at both the amino- and carboxy-terminal ends. Plasma collected 4 days prior to parasitechallenge was tested by ELISA and immunoblotting forreactivity with these proteins.

Nucleotide sequence accession number. The EMBL acces-sion number for P. falciparum Kl gpl90 is X03371. Thereare no other gene accession numbers applicable for any ofthe recombinant proteins used in this report.

RESULTSImmunoblot analysis of native gpl90. The apparent molec-

ular weights of the polypeptides in the affinity-purified nativegpl90 immunogen, as revealed by reactivity with endemic-area plasma, corresponded to those previously published(13, 15, 16, 30) for the gpl90 precursor and its processedproducts (Fig. 1). When reacted with a total parasite extract,the rabbit antiserum used to purify native gpl90 as well asthe monkey antisera obtained prior to challenge from mon-keys immunized with native gpl90 revealed polypeptideswith molecular weights expected for native gpl90 (Fig. 1).Monkey protection studies. The results of monkey protec-

tion studies are shown in Fig. 2. By 12 days after receivingparasites, the four control monkeys required drug treatment.Three of four monkeys which had been immunized withp190-3 also required drug treatment by 12 days, while theremaining animal cleared the parasites after having maxi-mum parasitemia of 10%. Two of four monkeys which hadbeen injected with p190-1 required drug treatment on day 14,while one cleared the parasites after 16 days, with maximumparasitemia of 4%, and one still had 1% parasitemia by day21, with maximum parasitemia of 13% over this time period.The group of animals that received native gpl90 had thelowest levels of parasitemia. Because of a neck infection,which was discovered following immunization, one animal inthis group did not receive the parasite challenge. Of the

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T

5 10 15 20 25

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20-

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5 10 15 20 25

p1 90-1 T

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native gpl90

A .-

0 5 10 15 20 25

Time after challenge (days)FIG. 2. Effect of immunization with recombinant (p190-1 or p190-3) or native (native gpl90) polypeptides on challenge with blood-stage

P. falciparum. Control monkeys received only Freund's complete adjuvant. T, drug therapy.

remaining three, one was removed from the experiment 8days after challenge because of a blood infection and fever;during this time, its maximum parasitemia was 1.1%,whereas parasitemias in controls varied between 4.3 and 9%.The effect of the infection on susceptibility to parasitechallenge is unknown. The other two monkeys had maxi-mum parasitemias of 0.53 and 0.45% during the course of theexperiment.Monkey antibody responses. (i) Reactivity with recombinant

proteins. Prechallenge antibody responses in ELISA to bothp190-1 and p190-3 were high in animals receiving either ofthese recombinant proteins (Table 1). Monkeys receivingnative gp190 also produced high antibody titers to p190-3 butsomewhat lower antibody titers to p190-1. There was noobvious relationship between ELISA titer and protection.An attempt was made to more precisely localize the areas

of gp190 which were recognized by prechallenge sera. Thevarious p190 recombinant proteins were tested in ELISAand immunoblotting and yielded, in general, equivalent

results (data not shown). Native gp190 elicited antibodyreactive with residues 123 to 302 in the three monkeys. Inaddition, serum from one monkey (Milo) reacted with resi-dues 384 to 595, while that from another (Patrik) reacted withresidues 595 to 897 and 1078 to 1251. Sera from the threegpl90 monkeys reacted with residues 34 to 595 and 1397 to1679.Each monkey receiving p190-3 or p190-1 produced anti-

body against residues 123 to 302, a sequence present in theoriginal antigen. In addition, each monkey injected withp190-3 produced antibody reactive with residues 1078 to1251, a sequence present in the original antigen.

It should be stressed that almost all of the antibody(>99%) elicited by p190-3 was specific for this parasitesequence. Nevertheless, three of the four monkeys immu-nized with p190-3 produced antibody reactive with thehexa-His sequence (data not shown).

(ii) Reactivity with parasites. In the p190-1 group, the twomonkeys with the highest titers against Palo Alto did not

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TABLE 1. Reactivity of monkey sera with p190-1 or p190-3

Elisa titer (103)% Maxi-

Antigen Monkey Preimmune Prechallenge mum para-sitemiaa

p190-1 p190-3 p190-1 p190-3

None Uno 2.3 0.9 5.8 2.3 TVasco 2.3 0.9 2.3 0.9 TWalo 2.3 2.3 2.3 0.9 TXeno 2.3 5.8 5.8 5.8 T

p190-1 Gallo 0.9 0.9 3,576 1,431 THardi 2.3 0.9 1,431 3,576 4.7Elmar 2.3 2.3 572 8,941 13.2Fabio 2.3 0.9 3,576 3,576 T

p190-3 Ingo 2.3 0.9 3,576 3,576 TJens 2.3 2.3 3,576 1,431 10Karel 2.3 0.9 3,576 8,940 TLaslo 0.9 0.4 1,431 572 T

Native gp190 Milo 2.3 0.9 229 3,576 0.45Nasin 2.3 0.9 92 1,430 1.1Patrik 0.9 0.4 92 572 0.53

a T, therapy.

require therapy, while the remaining two did. In the nativegpl90 group, in which the best protection was recorded, theanti-Palo Alto antibody responses were the highest. On theother hand, prechallenge antibody responses to parasites didnot demonstrate a relationship with protection in the p190-3group (Table 2). The only monkey (Jens) in this group whichdid not require drug treatment had the same low titer (640)against the parasite challenge strain (Palo Alto) as anothermonkey which did require drug treatment (Laslo). In eachcase, the fluorescence pattern was characteristic of theschizont stage. Considered collectively, monkeys in thethree experimental groups which recovered without drugtreatment had significantly higher antiparasite antibody titersthan did those requiring therapy (P = 0.082, two-tailedMann-Whitney test).

DISCUSSION

The purpose of the present experiments was to minimizeor eliminate the problems associated with strain variability inthe development of a vaccine against the blood stage of P.falciparum by use of recombinant protein vaccine candi-dates derived from relatively highly conserved areas ofgpl90. Unfortunately, although each protein conferred inone or two animals some degree of protection, neitherprotein elicited the extent of protection evoked by nativegpl90. In this regard, the results of the present study, inwhich excellent protection against heterologous challengewas provided by native gp190, confirm and extend theoriginal observations of Siddiqui et al. (32), who found thatnative gp190 from Palo Alto provided excellent protection inAotus monkeys against a homologous parasite challenge.The degree of protection in the present study was greaterthan that described by Hall et al. with a similar system (12).The basis for this difference is not known. It may be that theantigen preparations contained different amounts of thevarious gpl9(-derived polypeptides, leading to a markedeffect on the development of immunity. In another protec-tion study conducted with Aotus monkeys, of five monkeysimmunized with a Ki gpl90-derived fusion protein similar to

TABLE 2. Reactivity of monkey sera with parasites

Fluorescence titer% Maxi-

Antigen Monkey Preimmune Prechallenge mum par-asitemiaa

Kl Palo Alto Kl Palo Alto

None Uno <40 40 160 320 TVasco <40 <40 80 80 TWalo 80 80 80 80 TXeno 80 40 40 80 T

p190-1 Gallo <40 <40 5,120 640 THardi <40 <40 1,280 5,120 4.7Elmar <40 <40 5,120 10,240 13Fabio <40 <40 640 1,280 T

p190-3 Ingo <40 <40 1,280 1,280 TJens <40 40 1,280 640 10Karel <40 <40 10,240 5,120 TLaslo <40 <40 640 640 T

Native gpl90 Milo 80 40 20,480 40,960 0.45Nasin <40 40 40,960 10,240 1.1Patrik <40 40 40,960 40,960 0.53

a T, therapy.

p190-3 and challenged with isolate FVO (Ki type), two didnot require therapy, having maximum parasitemia levels of 6and 1.2%, while the remaining three did (14).The reason that p190-3 or p190-1 was not as effective as

native gp190 and the role, if any, played by differences in theamount of antigen administered are unknown. Perhaps nei-ther recombinant protein contains the correct structure(including primary, secondary, and/or tertiary structures) toelicit the type of effector lymphocytes and/or antibodyneeded for optimal protection. The use of a heterologouschallenge strain indicated that there was a sufficient degreeof similarity to gpl90 to impart marked protection. It isimportant to stress, however, that in addition to the rela-tively highly conserved regions of this protein, the entireN-terminal 25% of the Palo Alto gpl90 sequence is almostidentical to that of Kl, and these homologous areas mayhave played an important role in protection. A clear aim is toidentify a recombinant protein which duplicates this degreeof cross-protection. We note that an antigen purified fromparasites always has the risk of being contaminated withother parasite proteins, although we found no evidence ofthis in an immunoblotting analysis of prechallenge sera.The hexa-His sequence had a high affinity for Ni2+. It was

included in the recombinant proteins to facilitate purificationby Ni2+-chelate affinity chromatography. Immune recogni-tion of this sequence, as reflected by the presence ofantibody which is, of course, not parasite specific, is accept-able, provided that it does not interfere with protection. Asonly a very low amount of antibody (<1%) reactive with thehexa-His sequence was elicited, this sequence was clearlynot immunodominant; future experiments will be needed toestablish the effect of this sequence on protection.

Consistent with anti-gpl90 antibody being important forprotection in the present experiments, the highest antipara-site antibody titers were in the native gpl90 group, whichhad the best protection. Moreover, there was a significantrelationship between the monkeys in the experimentalgroups which did not require drug treatment and fluorescent-antibody titers to the challenge parasite. Both the amount ofantibody and its fine specificity may be crucial for protec-

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3502 ETLINGER ET AL.

tion. Potentially exemplifying this point was Hardi, whoseprechallenge antibody titers in ELISA for p190-1 or p190-3were similar to those of Gallo or Fabio; however, Hardi'santibody titer against the challenge parasite was higher and,of these three monkeys, Hardi was the only one not requir-ing drug treatment. The present results are consistent withthose of Hui and Siddiqui, who found a strong correlationbetween in vivo protection of Aotus monkeys by immuniza-tion with Pf195 (native gpl90) and inhibition of P. falciparumgrowth in vitro with sera from immunized monkeys (22).Moreover, in the P. yoelii mouse model, antibody to thegpl90 analog, a 230-kDa precursor protein, is also importantfor protection (7).An attempt was made to determine the regions of the Palo

Alto allele of gp190 which might be important for protection.Prechallenge sera from the three monkeys injected withnative gp190 and challenged with parasites each reacted withthe recombinant protein containing amino acids 123 to 302.This sequence of amino acids was also present in p190-3 andp190-1, neither of which was as efficacious. It may be thatthis sequence is, nevertheless, highly protective but that theantibody which is elicited by native gpl90 and which isreactive with amino acids 123 to 302 is distinct from thatwhich is elicited by p190-3 or p190-1.A second sequence against which all prechallenge sera

from the native gpl90 group of animals reacted containedamino acids 1397 to 1679. This recombinant protein, fromthe carboxy-terminal end of P. falciparum gp190, contains acysteine-rich area which has considerable homology with acysteine-rich area from the carboxy-terminal end of the P.yoelii 230-kDa precursor protein. Recombinant proteins con-taining the latter cysteine-rich domain have been found tocontain an epitope recognized by a monoclonal antibodywhich protects against infection by blood-stage P. yoelii, andit was suggested that this area may be similarly protective inP. falciparum malaria (2). Ongoing experiments are neededto establish this point. Recombinant fusion proteins contain-ing sequences which include this carboxy-terminal regionfrom P. falciparum have been tested in monkey protectionexperiments, and some protection has been noted. Thus,two of four Aotus monkeys immunized with such proteinsrequired therapy, while the other two had maximum para-sitemias of 2 and 4%; three of three control animals requiredtherapy (17). The lack of good protection with these proteinscould reflect the absence of the correct parasite native gpl90structure(s) and/or a reduction in efficacy because of thepresence of unrelated amino acids in the constructs. Addi-tionally, other portions of gpl90 may be necessary.

Results from malaria-endemic areas indicate that protec-tion against malaria is associated with antibody (and cellularproliferative responses) to the carboxy end of gpl90 as wellas with high levels of antibody reactive with a region near theamino end, 190 L (amino acid residues 147 to 321 of K1) (31).The present serological results in monkeys are consistentwith these observations in humans. Other results frommalaria-endemic areas point to the important role played bynonconserved-region sequences in protection (8). In anyevent, the results presented here indicate that precursorprotein gp190 and/or its processed products can provideexcellent protection against a heterologous challenge of P.falciparum in Saimiri monkeys; our aim is to identify theimportant protective sequence(s) and test its efficacy inhumans.

ACKNOWLEDGMENTSWe are grateful to Inez Bolliger, Marie-Francoise Girard, Karen

Hollander, Sabine Kappey, Catherine Karrer, Daniele Kronen-berger, and Bernd Poeschl for excellent technical assistance; BeatWipf for providing the bacterial cells; Ulrich Certa and Reiner Gentzfor initial cloning of p190-1 and p190-3; Richard Pink for discussionsand critical reading of the manuscript; Jana McBride for the p190prototype analysis of P. falciparum; and Klaus Frueh, MichaelMueller, and Hermann Bujard for supplying material for the pro-duction of recombinant proteins.

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of Plasmodium falciparum impair the development of protectiveimmunity against malaria. Parasite Immunol. 8:529-539.

2. Burns, J. M., W. R. Majarian, J. F. Young, T. M. Daly, andC. A. Long. 1989. A protective monoclonal antibody recognizesan epitope in the carboxy-terminal cysteine-rich domain in theprecursor of the major merozoite surface antigen of the rodentmalarial parasite, Plasmodium yoelii. J. Immunol. 143:2670-2676.

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7. Freeman, R. R., A. J. Trejdosiewicz, and G. A. M. Cross. 1980.Protective monoclonal antibodies recognizing stage-specificmerozoite antigens of a rodent malaria parasite. Nature (Lon-don) 284:366-368.

8. Fruh, K., H.-M. Muller, A. Crisanti, A. von Brunn, S. L.Hoffman, C. N. Oster, J. D. Chulay, M. Mugambi, H. Bujard,and J. A. Lyon. Unpublished data.

9. Gentz, R., U. Certa, B. Takacs, H. Matile, H. Doebeli, R. Pink,M. Mackay, N. Bone, and J. G. Scaife. 1988. Major surfaceantigen p190 of Plasmodium falciparum: detection of commonepitopes present in a variety of plasmodia isolates. EMBO J.7:225-230.

10. Goman, M., G. Langsley, J. Hyde, N. Yankovsky, W. Zolg, andJ. Scaife. 1982. The establishment of genomic DNA libraries forthe human malaria parasite Plasmodium falciparum and identi-fication of individual clones by hybridization. Mol. Biochem.Parasitol. 5:391-400.

11. Good, M. F., J. A. Berzofsky, and L. H. Miller. 1988. The T cellresponse to the malaria circumsporozoite protein: an immuno-logical approach to vaccine development. Annu. Rev. Immunol.6:663-688.

12. Hall, R., J. E. Hyde, M. Goman, D. L. Simmons, I. A. Hope, M.Mackay, J. Scaife, B. Merkli, R. Richle, and J. Stocker. 1984.Major surface antigen gene of a human malaria parasite clonedand expressed in bacteria. Nature (London) 311:379-382.

13. Hall, R., A. Osland, J. E. Hyde, D. L. Simmons, I. A. Hope, andJ. G. Scaife. 1984. Processing, polymorphism, and biologicalsignificance of p190, a major surface antigen of the erythrocyticforms of Plasmodium falciparum. Mol. Biochem. Parasitol.11:61-80.

14. Herrera, S. M., A. Herrera, B. L. Perlaza, Y. Burki, P. Caspers,H. Doebeli, D. Rotmann, and U. Certa. 1990. Immunization of

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16. Holder, A. A., and R. R. Freeman. 1984. The three majorantigens on the surface of Plasmodium falciparum merozoitesare derived from a single high molecular weight precursor. J.Exp. Med. 160:624-629.

17. Holder, A. A., R. R. Freeman, and S. C. Nicholls. 1988.Immunization against Plasmodium falciparum with recombinantpolypeptides produced in Escherichia coli. Parasite Immunol.10:607-617.

18. Holder, A. A., M. J. Lockyer, K. G. Odink, J. S. Sandhu, V.Riveros-Moreno, S. C. NichoUs, Y. HilIman, L. S. Davey,M. L.-V. Tizard, R. T. Schwartz, and R. R. Freeman. 1985.Primary structure of the precursor to the three major surfaceantigens of Plasmodium falciparum merozoites. Nature (Lon-don) 317:270-273.

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20. Howard, R. J., J. A. Lyon, C. L. Diggs, J. D. Haynes, J. H.Leech, J. W. Barnwell, S. B. Aley, M. Aikawa, and L. H. Miller.1984. Localization of the major Plasmodium falciparum glyco-protein on the surface of mature intraerythrocytic trophozoitesand schizonts. Mol. Biochem. Parasitol. 11:349-362.

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22. Hui, G. S. N., and W. A. Siddiqui. 1987. Serum from Pf195protected monkeys inhibit Plasmodium falciparum growth invitro. Exp. Parasitol. 64:519-522.

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