Proc. Natl. Acad. Sci. USAVol. 87, pp. 4017-4021, May 1990Medical Sciences

Immunization of Aotus monkeys with Plasmodium falciparumblood-stage recombinant proteins

(malaria/vaccine/recombinant antigens)

S6CRATES HERRERA*, MYRIAM A. HERRERA*, BLANCA L. PERLAZA*, YVONNE BURKIt, PATRICK CASPERSt,HEINZ DOBELIt, DANIEL ROTMANNt, AND ULRICH CERTAtt*Department of Microbiology, School of Health, Universidad del Valle, Cali, Colombia; and tCentral Research Units, F. Hoffmann-La Roche Ltd., CH-4002Basel, Switzerland

Communicated by William Trager, March 19, 1990 (receivedfor review December 7, 1989)

ABSTRACT The current spread of multidrug-resistantmalaria demands rapid vaccine development against the majorpathogen Plasmodium falkiparum. The high quantities of pro-tein required for a worldwide vaccination campaign selectrecombinant DNA technology as a practical approach forlarge-scale antigen production. We describe the vaccination ofAotus monkeys with two recombinant blood-stage antigens(recombinant p41 and 190N) that were considered as vaccinecandidates because parasite-derived antigen preparationscould protect susceptible monkeys from an otherwise lethalmalaria infection. In contrast to the natural antigen, recom-binant p41 protein (P. falciparum aldolase) could not protectmonkeys, although all animals seroconverted. 190N antigen, arecombinant protein containing conserved sequences of themajor merozoite surface antigen p190, protected two of fivemonkeys from critical levels ofinfection with the highly virulentFVO isolate of P. falckarum. However, the B- and T-cellresponses to 190N antigen were similar in protected andunprotected animals so that other unknown factors may con-tribute to protection. Higher purity or lack of protectiveepitopes or different structure of protective epitopes in therecombinant proteins might explain the better performance ofparasite-derived antigens in vaccination trials. The partialprotection obtained with 190N antigen suggests that this mol-ecule could contribute to a vaccine mixture against P. falci-parum.

The unicellular protozoan Plasmodium falciparum is themajor pathogen causing lethal malaria in man. The sporozoitestage of the parasite is transmitted by the bite of a femaleAnopheles mosquito, invades the liver of the host, anddevelops into a mature schizont. Subsequent release ofmerozoites into the blood stream starts the infectious cycle,which ends with the manifestation of clinical malaria symp-toms. Insecticide spraying briefly controlled the spread ofthedisease until resistant mosquito vectors developed. Morerecently, the worldwide spread of drug-resistant malaria haseliminated the hope of eradicating malaria by chemotherapy.The partial failure to control malaria by drugs explains theurgent necessity ofadditional means for malaria control, suchas vaccines.

Successful experimental immunization trials with irradi-ated sporozoites (1), blood-stage parasites (2), and later withpurified parasite antigens (3-5) demonstrated that P. falci-parum is vulnerable to immune attack in experimental infec-tions. The major coat protein of merozoites referred to aspl90, gp195, or PSA (polymorphic schizont antigen) is apolymorphic glycoprotein with Mr between 190,000 and205,000. Parasite-derived p190 and its processed products

purified from parasite cultures could protect Aotus monkeyscompletely from malaria, and therefore, recommends it as apotential vaccine candidate (5). Because of the problem ofantigen polymorphism (6, 7), two conserved regions of thep190 molecule (amino acid residues 146-312 and 1059-1196;ref. 8) were expressed in Escherichia coli as a single fusionprotein termed 190N (9). The use of strongly conservedregions of p190 reduces the risk of antigenic variation trig-gered by immunization. 190N antigen is recognized by sera ofhumans with acquired semiimmunity to malaria (9) andcontains B- and T-cell epitopes (10). These properties madeit an attractive subunit vaccine candidate based on theprotective natural p190 molecule (9, 10).The other recombinant protein included in this study is a

protein ofMr 41,000 (termed p41), which was identified as thealdolase of the parasite (4, 11). The parasite-derived antigenpreparation gave a marked degree of protection againstmalaria in immunized Saimiri monkeys (4). Probably forfunctional reasons, the entire polypeptide sequence is highlyconserved in all P. falciparum isolates analyzed (11). Thecentral function of aldolase for parasite metabolism makes itunlikely that mutants due to immunoselection will appearafter immunization. Successful expression of the entire poly-peptide in E. coli allowed us to vaccinate monkeys with theentire molecule (12). The sequence of the recombinant anti-gen lacks only five amino acid residues present in the naturalantigen. This lack does not affect the enzymatic or immuno-logical features of the recombinant antigen, which are virtu-ally identical to the natural protein (12).We describe the testing of both vaccine candidate recom-

binant proteins (rp4l and 190N) in Aotus monkeys. In con-trast to the natural antigens, rp4l gave no protection in twoindependent experiments and 190N protected two of fiveanimals from malaria after immunization and challenge withthe highly virulent FVO isolate of P. falciparum. Probableexplanations for the different results from experiments withrecombinant and parasite-derived antigens are discussed.

MATERIALS AND METHODSMonkeys. The monkeys (Aotus trivirgatus griseimembra)

were of Colombian origin and bred in the animal facilities ofthe Universidad del Valle (Cali, Colombia). Each experimen-tal group contained five animals matched for size and age.The monkeys had the karyotypes II and III, and each animalweighed -'800 g. No previous exposure to P. falciparum wasevident according to clinical history, antimalarial antibody,or peripheral blood mononuclear cell stimulation.

Antigens. The essentially full-length p41 protein expressedin E. coli, recombinant p41 (rp4l), and 190N were purified as

Abbreviations: rp4l, recombinant p41; PRBC, parasitized erythro-cytes (red blood cells); IFA, indirect immunofluorescence.tTo whom reprint requests should be addressed.

4017

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

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 8,

202

0

4018 Medical Sciences: Herrera et al.

described (8, 12). 190N antigen was further purified by an

additional SDS/gel purification step followed by overnightdialysis and lyophilization. Both antigens and the controlantigen (egg white lysozyme; Sigma) were dissolved in phos-phate-buffered saline (PBS) at 700 ,ug/ml. The antigen prep-

aration displayed a single band on SDS/polyacrylamide gelsdirectly before immunization (data not shown).

Immunization. Antigen was injected at day-0, -21, and -42ofthe experiment. At the same time the animals were bled forimmunological assays. Each dose consisted of 350 gg ofantigen dissolved in 500 p1L of PBS. Immediately beforeimmunization the antigen solution was mixed with 500 ,ul ofcomplete Freund's adjuvant for the first dose or with 500 ulof incomplete Freund's adjuvant for the booster injections.The sample was divided in four 250-pul aliquots and injecteds.c. into four different spots on the back of each animal.

Parasites. Three P. falciparum isolates, FUP Palo Alto(Uganda), FCB-1 (Colombia), and FVO (Vietnam) previ-ously adapted to Aotus monkeys were used. Parasite liquidnitrogen stocks were thawed and used directly to infect themonkeys.

Parasite Challenge. Fifteen days after the third immuniza-tion, all animals were challenged with 5 x 106 parasites of theFVO isolate of P. falciparum injected i.v. Parasitized eryth-rocytes (PRBC) were obtained from a monkey donor withexponential parasite growth. After challenge, the parasitemiawas monitored daily independently by us and the Cali MalariaCentre by both thin and thick smears stained with Giemsadye. Animals with 10% parasitemia were considered unpro-tected and immediately treated with Fansidar (Hoffmann-LaRoche). In one case (animal A17) Fansidar was given at6.5% parasitemia to save the animal, which showed severemalaria symptoms.

Determination of Protein Concentration. Protein concen-trations (antigen, PBRC) were determined colorimetricallyby using a commercial protein assay (Bio-Rad) and theprotocol supplied by the manufacturer (Bio-Rad, D-8000).

Analysis of Immune Response. Immune response in serawas maintained at day-0, after the first, second, and thirdimmunizations, and, in addition, 5 or 10 days after parasitechallenge. Antibody levels and specificities were determinedby indirect immunofluorescence (IFA), ELISA, and immu-noblotting.IFA. IFA was accomplished by using serial dilutions of

antisera in PBS and analyzed on multispot slide preparationsof FCB-1 parasites fixed with acetone (4).ELISA Assay. ELISA was done essentially as described by

Perrin et al. (4).Immunoblot. Immunoblot analysis was performed as de-

scribed (13). Each strip had one lane with a FCB-1 parasitelysate (30 ,ug of protein per lane) and two lanes with purifiedrp4l and 190N antigens (0.5 ,ug each). The monkey sera usedwere diluted 1/1000 in PBS. Antigen-antibody complexeswere detected with 125I-labeled protein G (Amersham) fol-lowed by autoradiography. Rabbit serum to rp4l or 190Nantigens (9, 11) was included as a positive control.Lymphocyte Proliferation Assay. Peripheral blood mono-

nuclear cells from each monkey were separated from hep-arinized blood obtained before the first and after the lastimmunization (day-42). Peripheral blood mononuclear cellswere purified by Ficoll-Paque density gradient under theprotocol of the supplier (Pharmacia). Cells were suspended at5 x 106 cells per ml in RPMI 1640 medium (GIBCO) supple-mented with 5% monkey B (-) serum, 2 mM L-glutamine, 25mM Hepes, penicillin at 100 units/ml, and streptomycin at100 ,ug/ml (GIBCO). Cells were cultured in triplicates of200-,ul aliquots in 96-well microtiter plates (Nunc) with either

a FCB-1 P. falciparum crude-antigen preparation (10 pug ofprotein per ml), an erythrocyte lysate (5 pug/ml), lysozyme at5 tkg/ml, rp4l antigen at 5 tkg/ml, or 190N antigen at 5 ,ug/ml.

Control plates were stimulated with Con A at 10 Ag/ml(Difco). Plates were incubated for 6 days at 370C in 5%C02/95% air. Cells were pulse-labeled during the final 24 hrof culture with 1 puCi (1 Ci = 37 GBq) of tritiated methyl-thymidine (specific activity 2 Ci/mM, Amersham). Cultureswere harvested onto glass-fiber filters by using an automateddevice, (miniMASH, Microbiological Associates) and pro-cessed for liquid scintillation counting.

Aldolase Inhibition Assay. Ten ng of rp41 aldolase wasmixed with 1 A.l of monkey serum, and inhibition of aldolaseactivity was determined colorimetrically by using a commer-cial kit (Sigma) according to the supplied protocol. Rabbitserum to rp41, that inhibited aldolase by 80%o under the sameconditions, served as a positive control, and preimmuneserum of the same animal (G574) was used as a negativecontrol.

Production of Rabbit Serum Against rp4i Antigen. A rabbit(G574) was immunized with one 200-iug dose of rp4l incomplete Freund's adjuvant and two 200-pg doses of rp4l inincomplete Freund's adjuvant (Sigma).

RESULTSBefore P. falciparum challenge we had established the im-mune status of the experimental animals. In particuar, wewere interested whether the immune system stinulatod withrecombinant proteins would interact with the natural antigenspresented by the parasite.Humoral Immune Response. Table 1 summarizes ie ma-

laria-specific B-cell response of all immunizeduuimalbeforeparasite challenge. Animals injected with PIfalc2parum-derived recombinant proteins 190N and rpl4sercmnweted.Chicken lysozyme was included as a nonrelated antigen in thecontrol group. All sera of monkeys immunized with rp41 or190N recognized the natural antigen on fixed parasites (IFA),in ELISA assays, and denatured in immunoblots (Table 1).The antibody titers against rp4l and 190N antigens are of the

Table 1. Prechallenge antibody titer in immizembAtliC&"ELISA titert

Monkey IFA* PRBC rp4l 190N ImmunoblotstAntigen rp4lA21 80 500 100.000 - +A17 80 1000 256.000 - +F6 320 1000 200.000 - +C3 320 1000 400.000 - +F27 1280 1000 256.000 - ++

Antigen 190NF71 320 1500 - 256.000 ++D73 80 500 - 100.000 +F48 320 500 - 100.000 +B12 20 500 - 128.000 ++B33 80 1000 - 16.000 +

Antigen LYSF104 - - - - -

C36 - - - - -

F56 - - - - -

Fill - - - - -

F67 - - - - -

LYS, Hen egg white lysozyme. -, Negative reaction in the assay.*IFA results are expressed as reciprocal end-point dilutions.tResults are expressed as the reciprocal of the dilution that yields anabsorption of 1.0 unit at 405 nm. PRBC is a lysate of FCB-1 P.falciparum isolate; rp4l and 190N are recombinant proteins ukd forcoating and immunization of monkeys.tTotal proteins of the FUP Palo Alto lysate were electrblaitd ontonitrocellulose membranes and incubated with a41GOW t~on ofeach monkey serum. +, Positive detection after overnight~expo-sure; + +, strong reactivity under the same conditions. -X

Proc. Natl. Acad. Sci. USA 87 (1990)

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 8,

202

0

Proc. Nadl. Acad. Sci. USA 87 (1990) 4019

same order of magnitude as in animals protected by immu-nization with the natural antigens (4, 5). Sera of semiimmuneindividuals from malaria endemic areas had similar ELISAvalues under the same assay conditions (PRBC: 1/10.000;rp41/1: 1000; 190N 1/2400).The p41 antigen had already been identified as the parasite

aldolase, an essential glycolytic enzyme located in the cyto-sol (11). We asked whether inhibition of the enzymaticactivity of p41 by antibodies contributes to protection. Wetherefore assayed aldolase activity in the presence of pre-challenge sera of all experimental animals. As expected, onlysera from the rp4l group could block the enzyme activity tothe same degree as a rabbit control serum raised againstparasite p41 (data not shown). A similar degree of aldolaseinhibition was measured with sera from protected monkeysimmunized with denatured or native parasite-derived aldo-lase (14).We thus assumed the B-cell response determined in four

independent assays to be sufficient for, at least, partialprotection.

Cellular Immune Response. Lack of a correlation betweenprotection and antibody titers in a previous and this trialsuggested that other immune mechanisms, such as a cyto-toxic T-cell response, could contribute to protection (4). We,therefore, measured T-cell stimulation in all experimentalanimals before immunization, after the second dose of anti-gen, and 10 days after parasite challenge (Fig. 1). Thepreimmune stimulation index with either pure antigen orPRBC as antigen had background levels in all animals tested.Prechallenge Con A-induced T-cell proliferation varied ex-tensively depending on the animal. Both recombinant anti-gens induced antigen-specific T-cell proliferation with rp4l,inducing significantly higher responses than 190N antigen

2cocmA rp~l lOON pROC comA rp~ll4ON pRMC comA rp41

200 F 104 C 36 F

150

100,

50 m0oo

(Fig. 1). PRBC antigen preparations stimulated T-cell prolif-eration in three of five rp4l-immunized animals, and the resthad background levels (Fig. 1). This result is probably relatedto the low amounts of specific antigen available for stimula-tion in parasite lysates. The T-cell stimulation index 10 daysafter the parasite challenge dropped significantly, even withCon A as stimulator-perhaps due to parasite invasion.

Selection of theP.faciparum Isolate for Challenge. To avoida primary immune response against the invading parasite,which could contribute to immunity, we wanted a P. falci-parum isolate that rapidly replicates in Colombian Aotusmonkeys. We thus infected three pairs of nonimmunizedmonkeys with the FUP Palo Alto (5 x 106 PRBC), FCB-1 (5x 106 PRBC), or FVO (2 x 106 PRBC) isolates of P. fal-ciparum. The course of infection was then followed by dailyblood examination. The FVO isolate, for example, reached1o parasitemia only 10 days after inoculation and requireddrug treatment, whereas the same parasitemia developedafter 20 days with FCB-1 and after 15 days with the FUP PaloAlto isolate of P. falciparum (data not shown). In the sameexperiment, we measured B-cell response against the invad-ing parasites during this test infection. None of the pairsresponded 10 days after infection in ELISA assays withPBRC as antigen. Twenty days after infection, however, allmonkeys seroconverted and developed significant titers ofparasite antibodies (1/80 against FVO, 1/80 against FCB-1,and 1/20 against FUP Palo Alto; data not shown). We thusselected FVO for the challenge infection.Course of Malaria Infection in Immunized Aotus Monkey.

Fig. 2 shows the development of parasitemia in the threegroups of animals immunized with either lysozyme (control)or the malaria vaccine candidates rp4l or 190N. As expectedfrom the test infection, high parasitemia started to develop

19ON pRBC conA rp 41 190 N pROC conA rp 41 190 N pROC

56 F 111 F 67

-3

250

200 A21 A17 F6 C3

X -'An i tg RS A

0

V'- 150.

x

E 100

0.O II

F 27

R-b -.

250

200 F71 D73 F48

150

100 _

p210 p210 p210 p210 p210 p210 p210 p210 p210 p210 p210 p210

B 12 B 33

p210 p210 p210 p210 p210 p210 p210 p210

FIG. 1. Development of T-cell response after immunization with lysozyme (Lys., Top), rp4l antigen (Middle), and 190N antigen (Bottom).T cells of each animal were stimulated before immunization (p), after the second immunization (no. 2), and 10 days (no. 10) after challenge witheither Con A (control, data set 1), rp4l (data set 2), 190N (data set 3), or PRBC (pRBC) (data set 4). The type of antigen for stimulation appears

at the top ofeach data set, and the animal code appears below. 3H counts incorporated are plotted on the y axis. Note that the response to controlstimulator Con A varies extensively depending on the animal. Values given are the average of triplicate measurements.

0.

z00)

Medical Sciences: Herrera et al.

__=C --o- -o- n

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 8,

202

0

4020 Medical Sciences: Herrera et al.

FIG. 2. Course of parasitemia in three groups of monkeys immunized with lysozyme (control group, Left), rp4l (Middle), or 190N (Right).Monkeys were challenged with the FVO isolate, and terminated curves indicate the point of chemotherapy in nonprotected animals. The twoanimals protected by 190N antigen (D73 and F71) had cleared the parasite at day-20 (data not shown). x axis shows days after challenge; animalcode numbers appear in the insets.

after day-10 in nonprotected animals. Development of para-sitemia in the control group and the animals immunized withrp41 was almost identical, although the mean parasitemia atday-9 was clearly lower than in the control group (Fig. 2). Atday-10 rapid parasite replication occurred in all animals oftherp4l group, which were rescued by chemotherapy. Becausethe results ofour vaccination trial deviate drastically from theresult obtained with a natural p41 antigen preparation, wedecided to repeat the experiment under the same conditionsto exclude experimental errors. However, results were iden-tical (data not shown).Two of five animals in the group immunized with 190N

antigen did not develop critical malaria symptoms; the peakparasitemia in these animals was 6% (D73) and 1.2% (F71).At day-20 these animals had completely cleared the parasites,and no blood-stage parasites were detected in the following8 weeks. Another animal (B12) had a delayed onset of criticalparasitemia. 190N antigen is thus significant as a p190-derived recombinant protein that partially protects Aotusmonkeys from malaria.

DISCUSSIONWe have tested two recombinant P. falciparum antigens(rp41 and 190N) as a malaria vaccine in the Aotus monkeyanimal model. The corresponding parasite-derived antigensprotected previously immunized monkeys from malaria (3-5). After immunization with the affinity-purified recombinantproteins, all animals seroconverted and developed similarparasite-specific antibody titers. The antibodies recognizedtheir natural equivalent in the native (IFA, ELISA) ordenatured conformation (immunoblot). In addition, bothantigens induced antigen-specific T-cell proliferation. Fur-thermore, rp4l antibodies inhibit the aldolase activity to thesame degree as in animals immunized with the natural protein(14). Despite this parasite-specific humoral and cellular re-sponse, rp41 antigen did not protect monkeys, but 190Nantigen induced partial malaria immunity.190N antigen contains :209%o of the natural p190 protein

sequence in an unnatural array, and thus epitopes requiredfor complete protection could be either missing or in anadverse configuration. This interpretation would help explain

why only two of five Aotus monkeys were protected againstmalaria infection. One protected animal (F71) had the highesttiters of p190 antibodies in all assays and the lowest peakparasitemia (1.2%). However, another animal (D73) hadclearly lower antibody levels, although it was protected (6%).The T-cell response in the protected animals was similar tothat of nonprotected monkeys (Fig. 1). This result maysuggest that sterile immunity requires stimulation of a yet-unidentified subset ofT cells by an epitope missing on 190Nantigen but present on the natural p190 molecule. Thisexplanation is consistent with the relatively poor stimulationobtained with PRBC as antigen.

Interpretation of the negative result obtained with rp4l,which contrasts with the result obtained with natural antigen,is more complicated (4). The following differences couldaccount for the different outcome of the two independentexperiments:

(i) rp4l antigen lacks the first five amino acids ofthe naturalantigen due to attachment ofan affinity sequence required forpurification; this pentapeptide could form an important epi-tope.

(ii) We cannot exclude that the p41 antigen preparationused in the previous trial (4) contained minor but highlyimmunogenic contaminants that contributed to protection.This explanation is unlikely because prechallenge sera ofprotected animals immunoprecipitated only one major bandof Mr 41,000 from parasite extracts (4).

(iii) The FVO isolate ofP. falciparum is more virulent thanFUP Palo Alto, requiring early control of parasite attack. InFig. 3 we compared postchallenge serum specificities ofp41-protected and unprotected animals immunized with therecombinant antigen. It is evident that the animals immunizedwith natural p41 antigen and challenged with FUP Palo Altodetected several additional parasite antigens. This crossre-activity was missing in the sera of the monkeys immunizedwith rp4l antigen and challenged with FVO. We have shownabove that a period of 20 days is sufficient for a primaryimmune response to the invading parasite. It is, therefore,difficult to determine whether the vaccine or the responseagainst the otherP.falciparum components protected againstmalaria in previous protection trials.

Proc. Natl. Acad. Sci. USA 87 (1990)

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 8,

202

0

MedicalSciences:Herreraetal.Proc. Natl. Acad. Sci. USA 87 (1990) 4021

A E

M 1 3 4

92-

69-

46

30-

14-

FIG. 3. Immunoblot analysis of postchallenge:and nonprotected monkeys. (A) Total parasite lysawith postchallenge sera of monkeys protected by infSDS/gel-purified natural p41 (4). Lane M, Mr x 11Same analysis with postchallenge sera from monIwith rp4l antigen. Only p41 antigen is detected aino

proteins 20 days after challenge with the FVO isolateat 1/1000 dilution. Codes for animals appear at topin A were adapted from Perrin et aL (4); codes in I

of the second rp4l protection trial.

Our results eliminate P. falciparum aldol,

vaccine candidate for malaria. The partial inhil

with 190N antigen, the ability to produce ar

amounts of the recombinant protein, and

properties of this protein suggest that 190N

become part of a malaria vaccine mixture th.

completed.

3 We thank Adriana Villegas, Hugo Blanco, and Pilar Crespo forMMM M

~~excellent technical assistance; Luc Perrin for design of the immuni-M6 14

M M zation shdl;and Bela Takacs for the anlssof immune serafrom monkeys protected by natural p41 antigen. We thank RichardPink for discussions and critical reading of the manuscript. We alsothank the Instituto Nacional. de Salud de Colombia for the gift of theAotus monkey colony. B.L.P. was supported by Research Grant1106-05-080-86 from Fondo Colombiano de Investigaciones Cientff-icas (COLCIENCIAS).

1. Clyde, D. F., McCarthy, V. C., Miller, R. M. & Woodward,W. E. (1975) Am. J. Trop. Med. 24, 397-401.

* e~~~i~ 2. Siddiqui, W. A. (1977) Science 197, 388-390.3. Perrin, L. H., Merkli, B., Loche, M., Chizzolini, C., Smart, J.

& Richle, R. (1984) J. Exp. Med. 160, 441-451.4. Perrin, L. H., Merkli, B., Gabra, S., Stocker, J. W., Chizzo-

lini, C. & Richle, R. (1985) J. Clin. Invest. 75, 1718-1721.5. Siddiqui, W. A., Tam, L. Q., Kramer, K. J., Hui, G. S. N.,

Case, S. E., Yamaga, K. M., Chang, S. P., Chen, E. B. T. &Kan, S.-C. (1987) Proc. Natl. Acad. Sci. USA 84, 3014-3018.

6. Tanabe, K., Mackay, M., Goman, M. & Scaife, J. G. (1987) J.Mol. Biol. 195, 273-287.

7. Certa, U., Rotmann, D., Matile, H. & Reber-Liske, R. (1987)EMOJ. 6, 4137-4142.

sera in protected 8. Mackay, M., Goman, M., Bone, N., Hyde, J. E., Scaife, J.- G.,ates were probed Certa, U., Stunnenberg, H. & Bujard, H. (1985) EMBO J. 4,amunization with 3823-3829.O0- markers. (B) 9. Gentz, R., Certa, U., Takacs, B., Matile, H., D6beli, H., Pink,ikeys immunized R., Mackay, M., Bone, N. & Scaife, J. G. (1988) EMBO J. 7,Ong P.falciparum 225-230.D.Sera were used 10. Sinigaglia, F., Takacs, B., Jacot, H., Matile, H., Pink, J. R. L.,of lanes. Codes Crisanti, A. & Bujard, H. (1988) J. Immunol. 140, 3568-3572.

3 are for animals 11. Certa, U., Ghersa, P., D~beli, H., Matile, H., Kocher, H. P.,Shrivastava, I. K., Shaw, A. & Perrin, L. H. (1988) Science240, 1036-1038.

ase (p41) as a 12. D6beli, H., Trzeciak, A., Gillessen, D., Matile, H., Shrivas-Lbition obtained tava, I. K., Perrin, L. H., Jakob, P. & Certa, U. (1990) MoI.id purify large 13Biochem. Parasitol., in press.thebilogicl 13.Certa, U., Bannwarth, W., Stuiber, D., Gentz, R., Lanzer, M.,

athbiolgincauld Le Grice, S., Guillot, F., Wendler, I., Hunsmann, G., Bujard,antigencouldH. & Mous, J. (1986) EMBO J. 5, 3051-3056.

at is yet to be 14. Shrivastava, I. K., Schmidt, M., Certa, U., Dobeli, H. &Perrin, L. H.- (1990) J. Immunol. 144, 1497-1503.

Medical Sciences: Herrera et A

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 8,

202

0