Clin. exp. Immunol. (1991) 84, 122-128 AixjNis 000991049100114M

Studies on the humoral immune response to a synthetic vaccineagainst Plasmodium falciparum malaria

M. SALCEDO. L. BARRETO, M. ROJAS, R MOYA. J. COTE & M. E. PATARROYOInstitute of Immunology. Hospital San Juan de Dios, Universidad Nacional de Colombia. Bogota. Colombia

(Accepted for publication 9 October 1990)

SUMMARY

A synthetic vaccine againsl the asexual blood stages of P. falciparum. the SPr66 synthetic hybridpolymer, composed of pcptides derived from three merozoite membrane proteins as v/ell as onepeptide from the sporozoite CS protein, has been developed by our group and tested in differentprotection assays in .4f>/».v monkeys as well as in human volunteers. This study evaluates the humoralimmune response induced by the SPf 66 protein vaccination in adult human volunteers from theColombian Pacific eoasl as follows: determination of specific IgG antibody levels against SPf 66 byFAST-ELISA after each immunization: analysis of antibody reactivity with P. fahiparum schizontlysates by immunoblots: and determination of the f>i )•;•?'-» parasite growih inhibition. A clear boostingeffect, dependent on time and dose, was observed in the antibody production kinetics. Theseantibodies also specifically reeognize three proteins of the P. falciparum schizont lysate correspond-ing to the molecular weights of the proteins from which the amino acid sequence was derived. Thesesera were also capable of markedly inhibiting in vitro parasite growth.

Keywords synthetic vaccines malaria vaecine human trial growth inhibitionantibody production

INTRODUCTION

ll is estimated that malaria caused by P. falciparum afflicts 234million people per year, killing 2-3 million, mainly children(Slurchler. 1989). To control this disease, several studies havebeen carried out in an eiTort to lind eHeclive immunoprophylac-tic methods (Nussenzweig & Nusscnzweig. 1984; Ballou ct ai.l987;Herringtoni?/a/., 1987; Jendoubi& PereiradaSilva, 1987;Collins et ai. 1988). Our group developed an effective syntheticvatxine againsl malarial parasites, the SPf 66 synthetic hybridprotein, directed mainly against the asexual blood stages of P.falciparum parasite, whieh in experimental trials has inducedprotective immunity in vaccinated human volunteers (Patar-royo (•/«/., 1988),

In order to expand the analysis of this vaccine we decided toperform two large-seale human trials to determine its safety,immunogenicity and protectivity and to defme the best immuni-zation schedule according to the kinetics of the immuneresponse againsl the synthetic hybrid molecule, the parasiteproteins and its growth inhibition. Quantitative determinationof igG-speeifie antibodies is important to determine the efficacyof a vaceine. since it has been suggested that these antibodies

Correspondence: Manuel Elkin Patarroyo, MD, Director, Institutode Immunologia. Hospital San Juan de Dios. Carrera lOa, Calle la,Bogota D.E.. Colombia.

block the erythrocyte invasion by merozoites or by affecting itssubsequent development (Mitchele et ai, 1976; Brown et al.,1982; Jepsen. 1983: Wahlin et ai. 1984).

Here we discuss the immunological aspects of these twolarge-scale human trials, in order to determine the relationshipbetween the IgG antibody production kinetics and the immuni-zation schedule. Other papers will discuss with the clinicalaspects of the trials. The kinetics of antibody production and itsduration, the specific reeognition ofthe parasite native proteinshy these antibodies as well as the in vitro P. falciparum growthinhibition were determined. The results of all these studies led usto design the most appropriate immunization schedule for thevaccine.

SUBJECTS AND METHODS

Human volunteersThree-hundred and ninety-nine healthy young male volunteers(age range 18-21 years) of the Colombian Military Forces, withal least 6 years of schooling were enrolled in the study; after acareful explanation of the project, written consent was obtainedfrom all participants. All the volunteers were young soldiersrecruited from the Colombian Pacific coast, who after 60 days ofmilitary induction stay in the malarial endemic area forapproximately 300 days of military service. The study popula-tion was composed of two groups with similar characteristics.

122

Serological studies in malaria vaccines 123

who performed their military service patrolling the same areas.The group named Tumaco A was made up of 193 men, of whom63 were randomly selected to be immunized with the syntheticvaccine and 130 were immunized with placebo, according to thesame protocol,, all on days 0, 20 and 220. The second group.Tumaco B, was enrolled 300 days later and included 206 people:122 were immunized with the vaccine and 84 with placebo ondays 0. 20 and 90. All the vaccinated volunteers receivedsubcutancously 2 mg of the hybrid synthetic protein SPf 66dissolved in saline solution and adsorbed to Al(OH}i in each ofthe three doses. The volunteers who received placebo wereinoculated with AI(OHh in saline soJution by the same route.None of the volunteers received prophylactic chemotherapyduring the study.

Serum samplesVenous blood was drawn to perform all the serological test. TheTumaco A volunteers were bled on days 0,19,37. 52, 98, 200 and240 after the firsi immunization. The Tumaco B group were bledon daysO, 14, 65, 110, 150 and 280 after the first immunization.The sera were separated, aliquoted. frozen immediately aftercollection and stored at —20 C in polypropylene vials withoutpreservative.

On certain occasions it was not possible to obtain venousblood samples from the same individuals who were on guardduty or patrolling in places other than the sample collection site.

SPf 66 antigenThe SPf 66 polymeric synthetic hybrid protein used forimmunization and for the FAST-ELISAs was produced at theInstituto de Immunologia by the Chemical Synthesis groupfollowing a published procedure (Merrifield, 1963; Barany &Merrifield, 1979). but using the multiple solid-phase synthesismethod described by Houghtcn (J985). The amino acidsequence ofthis protein in three-letter code is: Cys-Gly-Asp-Glu-Leu-Giu-Aia-Giu Thr-Gin-Asn-Val-Tyr Ata-Ala-Pro-Asn-Ala-Asn-P^o-Tyr-Ser-Leu-Phe-Gln-Lys-Glu-Lys-Met-Vai-Leu-Pro-Asn-Ala-Asn-P^o-Pro-Ala-Asn-Lys~Lys-Asn-A!a Gly-Cys-

After chemical synthesis of 50 g ofthe vaccine and extensivedialysis against pyrogen-free doubly distilled water, the puritywas checked by HPLC, SDS-FAGE, amino acid analysis andamino acid sequence. Toxicity, pyrogenicity, chemical composi-tion and stability tests were performed and repeated beforeusing this material as an immunogen in human volunteers.

Falcon assay screening test ELISA (FAST-ELISA)The FAST-ELISA, briefly described here, is a modification of adetailed method reported by Campbell et ai (1987), For theassay, 10/ig of the peptide per ml diluted in phosphate-bulferedsaline (PBS). pH 7 2, were placed in microtitre plates andincubated with shaking at room temperature for 150 min. Theantigen-coated lids were then washed by spraying with PBS-0-5% Tween 20 and rinsed with de-ionized water. These lidswere left to dry at room temperature and then stored in a jar withdessecant. After iheir thorough drying, the coated lids wereimmersed in microtitre wells containing the appropriate serumdilutions and incubated with shaking for 10 min at roomtemperature. The lids were then incubated with goat affinity-purified anti-human IgG peroxidasc conjugate, shaking for I h.and finaiiy incubated with the substrate for 5 min under the

same conditions. The lids were washed and rinsed after everystep. When the lids were removed, the optical densities weredetermined at a wavelength of 620 nm, using an ELISA plalereader.

The cut-ofi" point for the final tilrc determination was Ihemean of the optical densities of the preimmunc sera pius 3 s.d.

ParasitesThe FCB-2 P. fatciparum strain from Bogota, Colombia, wascultured in vitro with human group O Rh * erythrocytes using amodification (Zolg er ai. 1982) of the Trager & Jensen (1976)culture method.

Parasite tysate for Western MoilingLate stage schizonts from continuous P. falciparum cultures,,exhibiting 20% parasitaemia were collected, washed in sterilePBS and lysed in ^-l"/,, saponine solution with vigorousvortexing for 20 sec. The pellet was washed twice with largevolumes of PBS to remove hemoglobin and erythrocyte debris.Seven volumes of lysis buffer (5% SDS, I mM EDTA. 1 mMPMSF) were added to the pellet and then vortexing lor 10 min;the supernatant was further centrifuged at 22 50Oji; for 30 min.This lysafc was kcpl frozen in liquid nitrogen unlW use.

The non-infected red blood cell lysate was prepared in thesame way.

tmmunohtottingThe proteins were eiectrophoretically separated, transferred tonitrocelluiose paper (Towbin, Staehelin & Gordon, 1979) andincubated with pre-immune or immune serum at a 1/100dilution. The reaction was revealed with affinity-purified goatanti-human IgG alkaline phosphatase conjugate, the substratewas nitroblue tetrazolium (NBT) and BCI (5-bromo, 4-chIoro,3-indonJlphosphate) according lo the method oi' Blake ei ai(1984).

High, intermediate or low antibody response sera fromdifferent bleedings, obtained in 20 volunteers from Tumaco A,were tested.

In vitro growth inhibition a.\.sayThirty serum samples from the Tumaco A vaccinees, collectedon days 37,98 and 240 after the firsi immunization, were chosenlo evaluate the in vitro growth inhibition.

For this assay, cultures were synchronized with a singleLreatment with 5% sorbitol (Lambros & Vanderbcrg, 1979). Themature parasites (lute trophozoites and schizont.s) obtainedafter culluring for 72 h, were diluted with normal group O Rh'erythrocytes to a starting parasitaemia of 0-2% and adjusted toa 2% haematocrit with RPMI 1640 medium supplemented with25 mM HEPES. 1 mg/ml hypoxanthinc. 40 /ig/ml gentamycin.5 LJ/m! penicillin. 2 g/Y glucose and 5% NaHCO<.

Aliquols of these synchronized cultures (240/il) were placedin 96-well flat-bottomed mierocuiture piates to which 60 /d ofimmune or pre-immune sera were added to a final concentrationof 20%. Difiercnt concentrations of immune sera were used,diluting with human non-immune sera, while maintaining thesame final concentration. Sera, thoroughly diaiysed againstsaiine solution using 6000-8000 molecular weight exclusion cut-off dialysis mcmbrunes. were also assayed. Chloroquine wasused at a concentration of 016 fig/i^\ per well as a positivecontrol of m vitro growth inhibition. The plated cultures were

124 M. Salcedo et al.

incubated al 37 C in a 5% O:/5"/.. CO:/%"/<, N: gas mixture for72 h in order to see the serum effect during two cycles of parasitegrowth.

The cultures were resuspended at 24 h, and the medium waschanged at 48 h using fresh medium with the corresponding testserum. At the end of the incubation period, triplicate wells ofeach sample were pooled, centrifugated. and thin biood smearsstained with Gietnsa were prepared. The percentage parasitae-mia was microscopically assessed reading 1000 erythrocytes.Eighty per cent of the sera were assayed at least three times.

Growth inhibition over the 72-h period was determined bycomparing ihe percenlage parasilaemia in ihc cultures contain-ing immune sera with that of the cultures containing pre-inmiune sera, according to the following formula (Hui &Siddiqui, 1986):

% lnhibitioD=Parasitaemia (P - O) - (T - O)

Parasitaemia (P-O) xlOO

WhereP = pre-immune serum at 72 h; T = immune serum at 72 h; andO = starting of parasitaemia.

Normal non-immune human sera obtained from individualsfrom non-endemic malaria areas, were used in control cultures.

RESULTS

Antibody production kineticsWhen Ihc specilic anii-SPf 66 IgG levels were determined byFAST-ELISA. we found almost similar antibody productionkinetics in the sera of both groups according to the number ofimmiinizaiions. However, the response was not the same in ailindividuals. Hollowing the second immunization (Grumet.Mitchell &McDevitt, 1971; Grumet. 1972) three patterns oftheIgG antibody response againsl the synihelic polymer wereobserved using FAST-ELISA; group I. high responders. whoincreased their antibody litres between M6{}0 ;ind 1/25600;group II, intermediate responders. who increased their antibodytitres between 1/200 and 1/800; and group III, low responders.who did not respond to the synthetic peptide or in whom theantibody litres never increased above 1/100 (Fig. I),

The antibody production kinetics showed ihe followingpattern: a relevant increase in the specific IgG antibody levelsagainst SPf 66 was not observed after the first immunization inany of the vaccinees. However, 15-30 days after the second andthird immunizations, a very signiiicunt antibody increase wasobserved, which was higher after the third dose.

Antibody titres tend to plateau by day 30 after the secondand third dose, followed by a mild decrease in titre similar lothat seen for the classical IgG half-time response to a givenantigen. Antibodies remained in the peripheral blood for longperiods, up to day 180, indicating that these antibodies last forlong periods of time in the serum of the individuals. The fewvolunteers who reeeived two doses had lower antibody titresthan the ones wiih three doses.

Neither the pre-immune sera nor the sera from placebocontrols exhibited ant.ibodies against SPf 66.

Recognition of native P. falciparum proteinsWhen analysed by Western blotting, the sera from volunteersvaccinated two or three times SPf 66 had IgG antibodiesreaeting with the following proteins: 135 kD, 115 kD and 83 kD.

cleavage product from the precursor lo ihe major merozoitesurface antigens (PMMSA), the 195-kD protein. Some reactedwith the 35-kD and some weakly with the 55-kD protein. Theamino acid sequence of the vaccine was derived from thesemolecules, Pre-immune or placebo sera did not reacl with theseproteins, although a large number of P. falciparum proteins wasrecognized by sera from patients with a previous history ofmalaria (Fig. 2). A correlation was found between anti-SPf 66IgG tiires and the reactivity ofthe sera in the Western blot, andthis reaction was stronger in those volunteers who had threevaccinations and high antibody litres.

Absorption of Ihe sera with ihe synthetic hybrid polymer SPl'66 completely abolished all reactivity of these sera with theproteins in the Western blot and the ELiSA (data not shown),There was no reactivity between the sera and the red blood eellmembrane proteins at any time during the immunizations (datanol shown).

In vitro P, falciparum growth inhihitionParasite growth in the presenec of pre-immune sera was similarto IhLit of normal nonimmune human sera obtained fromindividuals coming from non-endemic malaria areas. Parasitae-mia increased approximately 45 limes during ihe 72 h period.Chloroquine inhibited parasile growth by 89"'l. and wasincluded as a growth inhibition positive control.

Table 1 shows in liiro parasite growlh inhibition using seracollected from the Tumaco A group at ditTercnt times during theimmunization process. Inhibition levels found in sera obtained37 and 98 days after the first immunization (17 and 78 days afterthe second dose) were relatively low. In contrast, the inhibitionpercentage of sera obtained 240 days after the lirst immuniza-lion (20 days after the third dose) increased dramatically formost of the sera from high and intermediate responders as wellas in the sera of some low lesponders.

Table 2 Includes the analysis of parasite growth inhibitionfrom a larger number of sera from the Tumaco A groupcollected 20 days after ibe third immunization and theirrespective antibody titres against SPf 66. Most of these serashow a parasite growth inhibition above 45"-;.. while a few showlow inhibition percentages. The aniibody titres ranged from 0 lo1/25600, Inhihilion levels ranging from 45"o lo IOO'!̂ i wereseenin sera with high antibody tiires (numbers 380, 353. 437, 424,460, 355 and 371), similar lo those seen with intermediate ones(numbers 422,429. 417. 357 and 492). but they were also presentin some sera with low antibody litres (numbers 326, 503 and368), Opposed to that, some sera with high or intermediateantibody tiires showed very little inhibilion of the parasitegrowth in vitro (sera 375,408. 477,473, 378 and 431). Sera fromindividuals injected wiih saline solution and Al(OH)i and bledon the same day did not have antibodies against SPf 66 and didnot inhibit in vitro growlh of P, fatciparum. This fael rules out anon-specific eifecl of the adjuvant used (Table 2).

It is important to note that parasite morphology was notaffected throughout the test, in sharp contrast with changesinduced by crisis form faetor described by Jen.'̂ en ft ai (1984).

No significant difference was seen hclween inhibition levelsproduced by nondialysed as compared lo the dialysed sera(Table 3). /*/u.vm(>(//w?H/(://t7/«/rHm growth inhibition was directlyproporlional to scrum coneentration, the inhibition decreasingproportionally to the concentration of immune serum added(Fig. 3), suggesting a concent ration-dependent effect.

Serological studies in malaria vaccines 125

2S600

IZ800

6400

3200

S 1600

I 800

400

200

100

0

Pre-immune 37 52 98 200 240

25600

12800

6400

3200

1600

BOO

400

aDo

KX)

0

b )

dose0

2na dose20

3rel dose220

19 37 52 96 200 240

25600

12800

6400

3200

1600

800

400

200

100

0

ic 1

1st close 2t\a dose Zt6 dose0 20 220Days ofterdrst immurniotion

19 37 52 98 200^ 240

1st dose 2nd dose0 20

ird dose220

25600

12800

6400

3200

I 1600

f 800

< AOO

200 -

100

d ) 25600

12800

6400

3200

1600

800

400

200

100

0pre-immune PI

25600

12800

6400

3200

1600

800

400

200

lOO

14 65 IIO 150 280

1st dose 2nd 3rddose dose

0 20 90

(f !

65 liO 150 260

1st dose 2nd 3radose dose

0 20 90

Doys of ter first immvnizaUon

I i:IIO [50 280

Is) dose 2nd 3rddose dose

0 20 90

Fig. I. Antibody kinetics of the vacinees during the immunization procedure. Each point represents the final titre of the antibodies.(a)Tumaco A high responders; (b)Tumaco A intermediate responders; (c)Tumaco A low responders; (d)Tumaco Bhigh responders;{e)Tumaco B intermediate responders; (f )Tumaco B low responders. The vut-ofl"point for the final titre detcrminaliun was ihc mean ofthe optical densities of the pre-immune sera + 3 s.d.

DISCUSSION

Previous work from our laboratory lias shown that the SPr66polymeric hybrid synthetic protein Induces protection in Aotusmonkeys as well as in humans against experimenlal challengewith the asexual blood forms of the P. fatcipurum malariaparasite indicating that vaccination with this synthetic moleculecan induce protective immunity against infection caused by thisparasite (Palarroyo et at., 1987, 1988; Rodriquez et al., 1990).This was the first vaccine against the asexual blood stages of theP. falciparum parasite for human use.

The present work was carried out to determine the kineticsof antibody produciion in accordance with the immunizationschedule, the duration of these antibodies in the serum, antibodyreactivity with native molecules, and the biological mechanismsof substances induced by vaccination with SPf 66 which couldproduce phenomena such as inhibition of erythrocyle invasion.

The IgG antibody production kinetics against the SPf 66polymeric molecule were established. These antibodiesincreased their levels at 15-30 days after the second and thirdimmunization, clearly showing a boosting effect after the thirddose. This effect was observed nol only in the increase of anti-SPf 66 IgG antibodies, but also in the parasite growth inhibitionwhich increased dramatically after the third dose. Reactivitywith the parasite native proteins, assessed by Western blotting,was also seen. It can be thus stated that these phenomena are theresult ofthe immunization with the synthetic vaccine since allthe prcimmune sera and the sera from the placebo platoon-matcvolunteers did not show any antibodies against the vaccine.Furthermore, their sera did not inhibit the parasite growth afterthe second or third dose of the vaccine nor were they reactivewith the parasite nulive molecules.

It was found that from day 25 today 30after thesecotid andthe third doses, levels of anti-SPf 66 antibodies have a tendency

126

553 437

M. Salcedo et al.

140 336 375 300

-« kD

35

Fig. 2. Western blotting showing the reactivity of IgG antibodies from scrum (diluted l/IOO)of six volunteere to P. falciparum lysale.Sera from individuals were taken before the immunization and 20 days after the third dose. The volunteer .138 had previous malariahistory. PI. pre-immuiic; 3td. after third immutiization.

to slowly decrease, following the kinetics of all the IgG half-lives. However, they can be detected approximately 180 daysafter the second (Tumaco A) and third doses (Tumaco B) inrelatively high titres.

SPf 66 immunization also Induces the recognition of parasitenative proteins, once again in a close correlation with theiintibody titres agiiinst SPf 66, as .seen in the immunoblots. Serafrom volunteers with high antibody titres specifically andstrongly recognized the proteins with molecular weights of135 kD. 115 kD. 83 kO (a cleavage product of the l95-kDprotein), 55 kD and 35 kD (in some eases) on P. falciparumlysates. This phenomenon was most prominent in the seraobtained after the third immunization. This reaction did notoccur in sera with lowantibody titres or pre-immune or placebosera.

Some individuals did not produce or increase their anti-bodies titres againsl SPI" 66 after the third dose. This could berelated to an ineffective presentation of the synthetic peptideassociated to certain HLA molecules or could indicate adefective recognition at the T cell level and bolh possibilities arebeing currently explored in our Institute.

Although the majority of the sera with high antibody titresagainst SPf 66 showed a great parasite growth inhibition andsome low antibody titres sera showed a low parasite growthinhibition, we did not find a direct correlation between iheseantibody titres and Ihe in ritro growth inhibition of ihe P.falciparum para.site. As shown in Table 2. some sera with lowantibody titres induced a high-growth inhibition, and some serawith high antibody titres occasionally exhibited poor parasilegrowth inhibition. This observation suggested that (i) variations

Serological studies in malaria vaccines 127

Table I. Correlation of in riiro growth inhibition of P.fiilcipanim and immunization schedule

TaMe 3. Effect of dialysis of sera on in vitrogrowth inhibition of P./atciparum FCB-2

Serum no.

380460424492431420503305

Classification

HHH1IILP

Inhibition (%)•

After37 days

O'O0 0

18 0260

2 00 07 00 0

After98 days

N.D.N.D.0 06 00 00 00 00 0

Afler240 days

77067 044073012 09-8

4500 0

• Days after the first immunization,H, high, I, intermediate, L, low responders; P. placebo.

Table 2. tn vitro growth inhibilion of P. falciparum FCB-2 and antibodytitres againsl SPf 66 in sera collected 240 days after the first immuniza-

tion

Serumno. Classificalior

Three doses380353437375408424460440355477473371378431422429417492420357326503415368

Placebos.1833133053444164.14

-iWW

w

n\\\iA-{

W

w

LLLL

ELISA

256002560025600128006400640032003200320016001600160016008008D0800800400400200100100

00

000000

Exp. 1

93H78734!75361439800

678

1265747371149060505

64

000000

Inhibition (%

Exp. 2

8060

IOO200

3572—98——810

- -73636773107174412180

000

——

Mean

87 + 974 ±1994±927 ±109±12

44±1367 ±8439800

74 ±104±6

1269±669±870±472±lI2±38l±i367 ±1046 ±6i 3 ± n7 2 ± l l

000000

Serum no.*

380355421

Inhibition (%)

Before dialysis

77098 057-0

Afler dialysis

750980520

* Sera were collected 240 days after thelirst immunization.

IOO

80

60

- 40

20

H, high, I. intermediate. L. low responders.

1/5 1/8 1/10 1/20Dilution of Immune serum in culture medium

Fig, 3. Effect of serum dilution on in viiro growth inhibilion ofP. fatciparum. Sera coHecled 240 days after idc llrsl immunization werediluted with human non-inimtiiie normal scrum al :i fmai concenirahonor20"/^i. A, scrum 437 (high responder}; • . serum 368 (low responder);O, serum 490 (intermediate responder); and n, scrum 380 (highresponder).

could exist in the idiotypic specificity ofthe dilTcreni antibodiesagainst the SPf 66 molecule and henee against the parasite;(ii) Ihal different immunoglobulin classes and/or IgG subclassesnot detecled by us by FAST-ELISA assays could be involved inthe Inbibilory response; or (iii) Ihat factors (difTerent from crisis-form faetor). other Ihan IgG. present in the sera eould inducethis phenomenon. Thus, the magnitude of the parasile growthinhibition eould depend on many factors, but clearly all thesefactors arc induced hy Ihe SPf 66 vaccine, .since none of the .seraof the volunteers inoculated with Al(OH), in saline solutionobtained on ihe same days and tested by Ibe same assays,showed any ofthe above described phenomena.

Growth inhibilion was also independent from serum dialy-sis, and (hese data support the idea (hat the/actor or factors thaiinduce this phenomena have a molecular weight higher than8 kD. and suggest also the possibility ihat this inhibition was notdue to anti-malarial drugs present in the serum.

128 M. Salcedo et al.

These resuiu confirm that Ihc SPf 66 malarial vaccine ishighly immunogenic in humans, and induces anli-SPf 66 IgGantibody production that recognizes ihc P. faUipanim specificproteins from which the amino acid sequence of the vaccine wasderived. These IgG antibodies, independently or joinlly withother serum factors induced by vaccination with SPf 66. inhibitin vitro growth of the parasite,

SPf 66 is an effective chemically synthetized vaccine againstmalaria for human use. Its action of mechanism could bemediated totally or partially by the humoral mechanismsdescribed here. We propose, based on the data shown above.that one possible schedule of vaccination for adequate antibodyproduction against the parasite is to immunize on days 0,20 and90 or 220. In our hands this immunization schedule produceshigh levels of antibodies in the vaeeinated individuals, and theseantibody titres persist for at least 180 days.

ACKNOWLEDGMENTS

This research has been supported by the Ministry of Public Health ofColombia, the German Leprosy Relief Association and OucidcntatPetroleum Company of Colombia, We would like to express ourgratiludLMo the volunteers olthf Military Forces of Colombia, ColonelJulio Cesar CAceres, LiuWncni l"ratn:isco Nune?.. MD, and LiutenenVCliira Zambrano. BSc. for their special collaboration during thesestudies and for their patience. MauHcio Calvo. Jose Luis Carvajal. DrOscar Noya and Dr Carlos Eduardo Tosta provided helpful suggestionsduring this study,

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