α-Difluoromethylornithine induces protective immunity in mice inoculated with Plasmodium berghei sporozoites

236 TRAN~ACXON~

OF THE ROYAL SIXIETY

OF

TROPICAL.MEDICINEAND HYGIENE(1986) 80, 236-239

a-Difluoromethylornithine inoculated with ‘Uniti

induces Plasmodium

protective berghei

immunity sporozoites

in mice

J. GILLET’, G. BONE’, P. LOWA’, J. CHARLIER~, A. M. RONA’ AND P. J. SCHECHTER~ de Parasitologic, Ecole de Sant.6 Publique, Universitk Catholique de Louvain, Clos Chapelle aux Champs, 1200 Brussels, Belgium; 2Merrell Dow Research Institute, Strasbourg, France Abstract

Mice inoculated weekly with Plasmodium berghei sporozoiteswhile under treatment with ar-difhtoromethylornithine(DFMO), an inhibitor of ornithine decarboxylase,developedprotective immunity againstsubsequentchallengewith this parasite.The percentageof mice protected was similarwhether DFMO alone(55%) or DFMO + chloroquine(65%) wasused.With chloroquine alone,only 12%of micewere protected. This protection waslong-lasting(at leastsix months).The immunity protectedagainstsporozoitesbut not againsterythrocytic form inoculation.It is suggested that this protection is induced by antigensreleasedfrom exoerythrocytic schizontswhosefurther developmentis inhibited by DFMO. Introduction

The eradicationof malariahasbeenattemptedfor years by mosquito control associatedor not with chemoprophylaxis.Unfortunately, this strategy has not reached the expected goal (BRUCE-CHWATT, 1980). With recent progress in immunology, an approachto malaria control by vaccination appears possible.Theoretically, a protective immunity could be obtainedagainsteachof the three successive stages of the asexualcycle of the plasmodiumin mammals: the sporozoitesinoculated by the mosquito, the exoerythrocytic (EE) schizontsin the liver and the erythrocytic schizontsor the free merozoitesin the blood. The inoculationof sporozoitesinducesan immunity which protects againsta reinfection by sporozoites in mice, in monkeys and even in man (see COCHRANE et a!., 1980,for review). The inoculation of merozoiteshkewiseinduces an immunity which preventsthe developmentof the parasitesin the blood (see SIDDIQUI, 1980, for review). The possibilitv of immunity againstsporozoitesand eryth&ytic forms is. thus. established.The difficultv in obtaininalame quantities of antigens from either sporozoiYes“or merozoites,however, prevents practical application. The purification and the cloning of the antigenic proteinsis, at present,the goal of many laboratories (ELLIS et al., 1983). To date, no protective immunity hasbeendemonstrated in mammalsagainstthe EE schizonts,which begin penetrationinto a liver cell a few minutesafter the mosquito infective bite. In the present paper, studyingPlasmodium berghei in mice,we report on the development of protective immunity induced by or-difluoromethylornithine. o-Difluoromethvlornithine(DFMO) isan irreversible, enzyme-activatedinhibitor of ornithine decarboxvlase (METCALF et al., 1978). This enzyme catalysesthe biosynthesisof putrescmeand its inhibition decreasesthe intracellular concentrationsof polyamines,essentialfactorsfor cell growth. DFMO retardscellulardivision, especiallyin thosecellswith a high rate of multiplication, suchastumour cellsor parasites(see SJOERDSMA & SCHECHTER, 1984, for

review). We recently studiedthe effect of DFMO on rodent malaria.This drug inhibits the EE schizogony of P. berghei in mice, but hasno action on erythrocytic parasites(GILLET et al., 1982). A 1% DFMO solutionin the drinking water preventsthe development of malaria in 92% of treated mice inoculated with sporozoites.If any exoerythrocytic schizontsdo mature, the young merozoitesreaching the blood multiply and produce a lethal parasitaemia. The presentpaperextendsthis work and demonstrates that treatment with DFMO during sporozoite inoculationprotectsmiceagainstsubsequent inoculation. Materials

and Methods

Animals P. berghei (Vincke & Lips, 1948), Anka strain, was maintained in TBssp mice, a strain in which P. bergti is always lethal (GILLET & HERMAN, 1974). These mice, bred in our laboratory, weighed about 20 g at the beginning of the experiments. The vector was Ampheles stephensi, reared at 28” in our insectary. They were fed on mice infected with P. berghei and, during the sporogonic cycle, were kept at 21°C. administration DFMO was given per OS,at a concentration of 1% in the drinking water. DFMO is rapidly excreted in mice with a plasma elimination half-life of only 23 minutes (Fozard, personal communication). During daylight hours, mice drink sparingly and the DFMO plasma concentration is likely to become very low. To distribute the drug intake more evenly throughout a 24-hour period, we placed the mice in a room at 27°C with controlled illumination, alternating 2-hour dark and 4-hour light periods. The mice adapted to this new lighting schedule after a week, increased their fluid intake to about 7 ml/day, and drank at more frequent intervals. The mean daily dose of DFMO for a 20 g mouse was about 3.5 g/kg. Chloroquine was given in the drinking water at a concentration of 50 mg/l, the mean daily dose being about 17 mgikg. Drug

Sporozoite

inoculation

Two methods of administration of sporozoites were used, intravenous (i.v.) inoculation and infection by the bite of infected mosquitoes. For i.v. inoculation, the sporozoites were harvested from anopheles fed on infected mice 18 days before. Wings, legs and abdomen of anopheles were

J. GII.LE?

tion of 10,000 sporozoites. As controls, naive mice were similarlv inoculated and alwavs became infected from the 4th day after challenge. All the animals were examined for parasitaemia every day in the first week after challenge and thereafter on alternate days. After 21 days, mice not demonstrating parasitaemia were considered protected.

discarded. Head and thorax were ground in cool TC 199 medium, first with mortar and pestle, then with a glass tissue grinder. The sporozoite suspension was centrifuged at low speed to separate the coarse tissue fragments and purified through a biphasic density gradient as described by BEAUDOUIN et al. (1977). This sporozoite suspension still contained insect tissue which acted as allergens. Thus, after two or three successiveinoculations, anaphylactic shock and death were often observed. To eliminate the loss of animals from anaphylaxis,, we also used the natural method of malaria transmissron, i.e., the bite of infective mosquitoes. Six mice, with shaved abdomen and fastened on their backs, were left for one hour in a cage containing about 200 anopheles infected 18 days before. The number of sporozoites received by each mouse was unknown, but this natural method of transmission delivered only mature sporozoites and proved quite reliable (during a one-year period, 830 mice were exposed to the bite of infected mosquitoes; 809 developed a parasitaemia and only 21 (2.5%) remained

Results Three treatment regimens were used in different groups of mice.DFMO alonewasadministeredto one group. However, in some mice receiving DFMO, EE

schizontsmature and the merozoitesreaching the blood cause a lethal parasitaemia(GILLET et al., 1982).This couldoccur after eachinoculationand, to eradicatethis parasitaemia,we addedchloroquine to DFMO in a secondgroup of mice. The third group receivedchloroquinealoneto control for its effectson subsequentchallenges. Of the 16 mice treated with DFMO alone and inoculatedintravenously twice with 10,000sporozoites, nine did not develop parasitaemia(Table I). When challenged28 days following the last inoculation, five of these nine were protected. A total of 113 mice were inoculated while under treatment with the combination of DFMO plus chloroquine.Of these,73 (65%)wereprotectedwhen challenged28 days later, a percentagesimilarto that

negative).

Immuniaation procedure Over a two-to three-week period the mice received two to three intravenous inoculations with 10,000 sporozoites or were infected by three infective mosquito bites. During this immunization period, the mice were treated either with DFMO, with both. DFMO and chloroquine, or with chloroquine alone. Drug administration always began five days before the first inoculation and was continued until eight days after the last. Challenge

found with DFMO

infection

I-Influence

of drugs on the immunization Protection

Immunization orocedure

survived

the challenge.

of mice by sporozoites after challenge* DFMO+

chloroquine

DFMO Protected

chloroquine

%

Protected Challenged

%

Challenged Sporozoite

alone.

In contrast, of 50 mice treated with chloroquine alone during the inoculation period, only six (12%)

The mice were challenged 28 days after the last sporozoite administration, i.e., 20 days after the cessation of drug administration. Challenge was always done by i.v. inoculaTable

237

et al.

Protected Challenged

%

inoculations

2 i.v. inoculations 3 i.v. inoculations Mosquito bites X 3

519 -

55 -

24140 14122 35151

2:

4i34

12

69

2116

13

Total

519

55

731113

65

019

0

0138

0

6150 o/30

12 0

Controls

total

*The micechallengedwerethosenot demonstratingparasitaemia after the immunizationperiod. This represented 9 of 16 mice inoculated in the DFMO group; there was no parasitaemiaamong the mice treated with DFMO + chloroquine or with chloroquine alone. Table II-Duration of the immunity shown to be protected at 28 days

Immunization procedure 2 i.v. inoculations 3 mosquito bites

Controls

-

total

induced

by combined

treatment

Time before rechallenge

(days) 85 72 146 188

with DFMO

and chloroquine

in mice

Protected Challenged

16117 33134 10111

94 97 91

619

67

O/26

0

.___

238

DFMO

AND

PROTECTIVE

IMMUNITY

Parallel control mice (without treatment or previous inoculation) were run with each challenge experiment.In eachcase,all control micesuccumbed. To assesswhether the immunity produced was durable, mice shown to be protected after a first challengewere rechallengedwith i.v. sporozoitesat various intervals up to six months later. A high proportion of mice resistedthe rechallenges(Table II). To determine stage specificity of the protective immunity produced,a group of 16mice shownto be protectedagainsti.v. sporozoitechallengewereinoculated with 2,000blood parasites.All micedevelopeda lethal parasitaemia. Discussion

Theseresultsdemonstratethat sporozoiteinoculation of mice during treatment with the polyamine biosynthesisinhibitor, DFMO, induces protection againstsubsequentsporozoitechallenge.This protec&n is long&sting and appearsto be stagespecific. DFMO is not an invariable inhibitor of the EE schizogony.In somemiceEE schizontsdo eventuallv mature. The reasonfor this incompleteprotection by DFMO in mice is not clear but mav be due to irregular patternsof drinking behaviour complicated by a short plasmaeliminationhalf-life for DFMO in this species.However, even when DFMO, 1250 mg/kg, wasinjectedintraperitoneallyevery two hours during the entire lengthof the EE cycle, i.e., 48 hours in thismodel, somemicestill developedparasitaemia. To compensatefor the parasitaemiaseenin some mice despitethe DFMO treatment, we treated mice with chloroquine during the immunization period. We assumed that chloroquinehadno actionon the EE cycle and no effect on the developmentof immunity sinceno evidenceof acquiredimmunity in man after someyears of chloroquine prophylaxis in hyperendemic areashas beenfound (COHEN. 1979).On the other hand, antibodiesagainst P. &zlcip&m have beendetectedin seracollectedfrom individualsliving in The Gambia (NARDIN et aE., 1979). Further, a protective immunity hasbeeninducedexperimentally in rats (VERHAVE,1975)and in mice (BEAUDOUIN et aE., 1977;ORJIH et al., 1982)by repeatedsporozoite inoculationswith simultaneouschloroquineadministration. It was, therefore, essentialto evaluate the respectiverolesplayedby DFMO and by chloroquine in the protection obtained in our experiments.With chloroquinetreatment alone,we observedonly 12% protection in our TBrsv mice. This protection rate is much lower than that describedby BEAUDOUIN et al. (1977) and by ORJIH et al. (1982) studying NIH/ NMRI mice and A/J mice respectively. This discrepancy may be due to various causes;the mostlikely being the hinher receotivitv of the TBFIP mice to P. berg&i, Anki strain. *WhenDFMO and‘chloroquine treatmentswere combined,the rate of protection of mice aaainstsnorozoitechallengewassimilarto that for a&nals treated with DFMO alone. From what is known aboutthe actionof DFMO at eachstageof the plasmodiumcycle, onecanspeculate aboutthe mechanism of the immunity inducedby this compound. Following inoculation, the sporozoites remainfor a short time, probablya matter of minutes, in the blood beforeenteringthe liver. DFMO, which inhibits only dividing cells, is unlikely to have any

IN

P. berghei INOCULATED

MICE

effect on the sporozoitesduring this brief stay in blood. SinceDFMO hasno actionon the erythrocytic cycle (GILLET et al., 1982),any parasitesubsequently reaching the blood can lead to lethal parasitaemia. Thus, mice treated with DFMO aloneand surviving have never harboured parasitesin their blood and antigensresponsiblefor immunity could not have come from blood stage parasites.It is probable, therefore, that the protective immunity induced by DFMO originatesin the EE schizonts.The affected schizontsprobably do not maturein the presenceof DFMO and may eventually liberate antigens. In preliminary experimentswe attempted to demonstrate, by immunofluoresence,the presenceof specificantibodiesin the seraof mice treated with DFMO olus chloroauine durina the immunization period. using theses&a, we obtaikeda muchstronger fluorescencewith 48-hour-old exoerythrocytic schizonts thanwith sporozoitesor bloodforms. TheseEE forms did not react with antiserapreparedagainst sporozoitesor blood forms. The developmentof immunity in other suborders of the Coccidia following DFMO treatment also suggestan actionat the EE schizontstage.HANSON et al. (1982) obtained a protective immunity against coccidiosisin chickensinfected with Eimeria tenella and treated with DFMO. In the Eimeriidea, the sporozoitesareliberatedinto the lumenof the gut and penetrate the epithelial cells without entering the circulation. There is no phaseof developmentin the blood. The schizogony which takes place in the epithelial cells of the intestine is analogousto the hepatic EE schizogony of the Haemosporidiidea (Plasmodium).We believe that in both casesthe protectioninducedby DFMO proceedsfrom antigens, liberatedbv the slow lvsis of the abortive schizonts. If our hypothesisis valid, it suggests the possibility of a new approachto immunizationin hyperendemic malariousareas.While individuals are under treatment with an appropriate agent which arrests EE schizogony,the mosquitocould act asthe vaccinator and lysis of the schizontswith releaseof the antigen could impart protection againstsubsequentinoculations. Acknowledgements We appreciate the services of G. Mattucilli, in chargeof the insectary,a key elementof this research. References Beaudoin,R. L., Strome,C. P. A., Mitchell, F. & Tubergen,T. A. (1977).Plasmodium berghk: imsnunizationof miceagainst theANKA strainusingtheunaltered sporozoite asanantigen.Experimental Parasitology, 42, l-5

Bruce-Chwatt, L. J. (1980).Essential Malariology. London: Heinemann, pp. 280-293. Cochrane,A. H., Nussenzweig, R. S. & Nardin, E. H. (1980).

Immunization

against

sporozoites.

In: Malaria.

Kreier. 1. P. (Editor). New York: Academic Press, pp. 163-202: Cohen, S. (1979). Immunity to malaria. Proceedings of the Royal Society of London, 203, 323-345. Ellis, J., Osaki, L. S., Gwadz, R. W., Cochrane, A. n., Nussenzweig, V., Nussenzweig, R. S. & Godson, G. N. (1983). Cloning and expression in E. coli of malaria sporozoite surface antigen gene from Plasmodium knowlesi. Nature,

302,

536-538.

J.

GILLET

Gillet, J. & Herman, F. (1974). Recherches sur le paludisme congenital de la souris avec PZarmodium be&i. Passage transplacentaire du parasite et son evolution. Bulletin of the World Health Organization, 51, 385-398. Gillet, J., Bone, G. & Herman, F. (1982). Inhibitory action of a-difluoromethylornithine on rodent malaria (Plasmadium berghet]. Transactions of the Royal Society of Tropical Medicine and Hygiene, 76, 776-777. Hanson, W. E., Bradford, M. M., Chapman Jr. W. L., Waits, V. B., McCann, P. P. & Sjoerdsma, A. (1982). o-Difluoromethylomithine: A promising lead for preventive chemotherapy for coccidiosis. American Jourd of Veterinary Research, 43, 1651-1653. Metcalf, B. W., Bey, P., Danzin, C., Jung, M. J:, Casara, P. & Vevert, J. P. (1978). Catalytic irreversible mhibition of mammalian ornithine decarboxylase (EC 4.1.1.17) by substrate and product analogous. 3ournal of the American Chemical Society, 100,2551-2553. Nardin, E. H., Nussenzweig, R. S., McGregor! I. A. & Bryan, J. H. (1979). Antisporozoite antibodies. Their frequent occurrence in individuals living in area of hyperendemic malaria. Science, 206, 597-599. Orjih, A. Il., Cochrane, A. H. & Nussenzweig, R. (1982).

et

al.

239

Comparative studies on the immunogenicity of infective and attenuated sporozoites of Plasmodium berghei. Transactions of the Royal Society of Tropical Medicine and Hygiene, 76, 57-61. Siddiqui, W. A. (1980). Immunization against asexual , blood-inhibiting stages of Plasmodium. In: Malaria. Kreier, J. P. (Editor). New York: Academic Press, pp. 231-262. Sjoerdsma, A. & Schechter, P. J. (1984). Chemotherapeutic implications of polyamine biosynthesis inhibition. CZinical Pharmacology ana’ Therapeutics, 35, 287-300. Verhave, J. P. (1975). Immunization wtth sporozoites: an experimental study of Plasmodium berghei malaria. Thesis, Katholieke Universiteit Nijmegen, The Netherlands, 121 pp. Vincke, I. H. & Lips, M. (1948). Un nouveau plasmodium de rongeur sauvage du Congo: Plasmodium berghei. Annales de la Socit% Belge de Midecine Tropicale, 28, 97.

Accepted

fm

publication

4th Grundy Memorial Lecture Wednesday Roval

Armv

24th September Medical

College,

1986 Millbank,

London SWl- . ‘Ecology of Tropical Disease’ Speaker: Professor H. M. J. Gilles, Liverpool School of Tropical Medicine

Medical Entomologists’Annual Dinner RAMC

Wednesday Headquarters

24th September Mess, Millbank,

1986 London

Details of both events from: Dr. N. R. H. Burgess, Royal Army Medical College, Millbank, London SWll? 4RJ. Tel: 01-834-9060, Extension 229

SW1

4th

February,

1985