Current considerations in vaccination of humans against malaria: Use of attenuated sporozoites in the immunization of human volunteers against falciparum malaria*

t&lenn of she World Hea.rh Organzauuor, 57 (Suppi. 1): 261-265 k1979) Use of attenuated sporozoites in the immunization of human volunteers against falciparum malaria* K. H. RIECKMANN,I R. L. BEAUDOIN,2 J. S. CASSELLS,; & K. W. SELL Three human volunteers were successfully protected against sporozoite challenge by immunization with attenuated sporozoites ofthe Tameniestrain ofPlasmodium falciparum from Ethiopia. The immunizing sporozoites were attenuated by exposing infected Anopheles stephensi mosquiros to X-rays at a dose ofat least 120 Gy (12 000 rad). These ir- radiated, infected mosquitos were allowed to feed upon volunteers, thereby inoculating sporozoites into their blood stream. During the 10- to 38-week period of immunization, volunteers were exposed 6-8 times, at irregular intervals, to a total of 440-987 irradiated infected mosquitos. Protection against challenge by nonirradiated infected mosquitos lasted for at least 8 weeks, but not 16 weeks, after the last immunization with irradiated sporozoites. By contrast, volunteers who were exposed to a total of200 orfewer irradiated infected nmosquitos on 2-4 occasions were notprotected upon challenge. Immunization by a sufficienr nutnber ofirradiated mosquitos infected with the chloroquine-sensitive Tamenie strainfrom Ethiopia also protected against challenge with the chloroquine-resistant Marks strain of P. falciparum from Viet N,Jam. The results obtained in these studies suggest that immunization with attenuated sporozoites may be a useful method of protecting small groups ofnonimmune individuals living in endemic areas. Thesefindings should encourage further efforts to develop a sporozoite vaccine against humnian malaria. In view of the encouraging results obtained by im- munizing mice with irradiated sporozoites of Plas- modium berghei (1), a collaborative study was in- itiated in 1971 between investigators at Rush-Presby- terian-St Luke's Medical Center, Chicago, Illinois and the Naval Medical Research Institute (NMRI), Bethesda, Maryland, to determine whether protection could also be induced in human volunteers by expos- ing them to irradiated sporozoites of Plasinodium falciparum. As it was deemed ethically unacceptable to inoculate crude suspensions of sporozoites into volunteers, exposure to sporozoites was achieved by 'From a joint malaria project bctL. cen the Jr:Ir.unopar:3sito]ogy Department, Naval Medicai Researca Institute, Betnesda, Maryland and Rush-Presbyterian-St Luke's Mfedical Center, Chicago, Ilir.ois This wvork was supported by the Na' a! MJcdical Resea:ch and De- velopment Command, Work Un-L No MN5lr.524 009.0031 andONR Contract No. N00014-72-A-0288-0001. The opirions or assertions contained herein are the private ones of lt authors and are nOL to be consiued as official cr reflecLng the vie.s of the US Na.-y or !he naval service at large. I Head, Division of Tropical aad Geographic MIedicine. Depart- ment ol Medicine, University of New Mexico, JAbaquerque, NM 87106, USA. ' Head, Malaria Divisioni, Department of Immunoparasitoiogy, Naval Medical Research AisLitu c, B-etheda. MD 20014, USA. I Director, Clinical Services. Naval Regional Mcdical Center, San Diego, CA 92130, USA I Scientific Director, National Innirute of Allcrgy and lnfec;ious Diseases, National lnsLitutes of Health, BeLhesda, MID 20205, USA. allowing participants in these studies to be bitten by irradiated infected mosquitos. It was realized, of course, that only a small proportion of sporozoites in the saliVary glands would be released by mosquitos, that sporozoite inocula would probably be consider- ably lower than those used in previous animal exper- iments, and that the quantity of immunizing antigen would not be known. On the other hand, the results of animal experiments were so promising that an attempt was made to duplicate these results with P.faciparurn in man. The studies were carried out at the Stateville Cor- rectional Center, Joliet, Illinois. Since human beings had tiever before received irradiated sporozoites, 5 in%estigators at NNIRI, including 2 of the authors of this paper, volunteered to be exposed to irradiated in- fected mosquitos in order to establish the safety of this procedure. They were bitten by several hundred irradiated infected or noninfected mosquitos once a week for a period of 6 weeks. Thorough clinical and laboratory examinations over a period of several months failed to show any es idence of adverse side- effects to the sporozoites, although local reactions to the mosquito biLes were obsenred in the case of 1 volunteer. After completion of these preliminary studies, investigations involving inmate volunteers were initiaTed to ascertain whether irradiated sporo- zoites couid confer protection against human falci- 391 -261- K. H. RWECKMANN ET AL. parum malaria. The results of these studies, obtained between 1972 and 1975, are described in this report. NATERIALS AND METHODS These studies involved the participation of 136 inmates of the Stateville Correctional Center who had volunteered for the programme. Volunteers were healthy men between 21 and 40 years of age who had no previous exposure to malaria. Detailed infor- mation about the study and its potential risks was given to each individual and the voluntary nature of participation, including the right to withdraw at any time, was emphasized. The studies were approved by the Human Investigation Committee of Rush-Presby- terian-St Luke's Medical Center and they were con- ducted according to ethical and medical procedures established previously (2). In these studies, 84 volunteers participated as gametocyte donors. They were usually infected with the Tamenie strain (Ethiopia) of chloroquine-sensitive P.falciparum. In some instances, the Marks strain (Viet Nam) of chloroquine-resistant P.fakcparum or the Chesson strain of P. vivax was used. Infections were induced through the bites of infective mosquitos or by intravenous inoculation of small aliquots of parasitized blood. Thick blood fllms were prepared at least once a day, stained with Giemsa stain, and ex- amined by the method of Earle & Perez (3) for asexual forms and for gametocytes. Participants were admitted to the malaria ward on the same day that they developed patent parasitaemia or clinical symp- toms and they were kept under close medical super- vision throughout the entire duration of the study. Early in the clinical course of malaria, gametocyte donors received subcurative doses of chloroquine and quinine to relieve symptoms and to prevent the occur- rence of high levels of parasitaemia. Although sup- pression of asexual parasites was essential for the safety and comfort of participants, it often prevented the subsequent development of an adequate number of gametocytes for infecting mosquitos. Conse- quently, only about one-third of the men developed a gametocytaemia capable of inducing heavy sporo- zoite infections. The mosquitos used for transmission of sporozoites were Anopheles stephensi reared at the Stateville in- sectary. After gametocyte donors had developed a parasite count of at least 100 gametocytes per mm3 of blood, mosquitos were allowed to bite them. En- gorged mosquitos were then returned to the insectary in a holding cage. Six days later, 5 mosquitos from the cage were examined for the presence of oocysts. On each of days 1O, 12, 14, and 16, the salivary glands of 5 additional mosquitos were examined for the pres- ence of sporozoites. Batches of mosquitos that were beavily infected (at least 25% of mosquitos with more than 1000 sporozoites per mosquito) were irradiated between 12 and 18 days after their blood meal. Irradiation of infected and noninfected mosquitos was performed at the Radiation Physics Section of Rush-Presbyterian-St Luke's Medical Center by means of a Westinghouse 220 kV (peak) ortho voltage X-ray machine. The tuibe was operated at a peak vol- tage of220 kV and 18 mA and the quality of the beam was dcfined by use of a 0.5-mm copper filter. The dis- tance between the surface of the sample and the target was 10.0 cm and the exposure rate was 9.1 x 103 C/(kg s) (2100 R/niin). The exposure time was deter- mined by measurement with a Victoreen R meter prior to the exposure of the sample and, in addition, a lithium fluoride disc was used to confirm the mini- mum dosage of 120 Gy (12 000 rad) for each sample irradiated. Mosquitos were irradiated in a plastic container measuring 1.0 cm in height and 9.4 cm in diameter. Back-scatter material was provided by placing the container on 13.0 cm of water. After X- irradiation delivering a minimum of 120 Gy, mos- quitos were transported back to the malaria ward and, within approximately 60 miR after irradiation, they were allowed to bite the appropriate participants in these studies. The actual immunization studies involved the par- ticipation of 52 volunteers. Eleven men werc bitten by irradiated infected mosquitos. The other 41 men were bitten either by irradiated noninfected mosquitos or by a few nonirradiated infected mosquitos (see below). The number of mosquitos per exposure varied from 7 to 500. Saline infusions were started in volunteers before each exposure to mosquitos in case rapid intravenous administration of drugs was re- quired to counteract allergic reactions to mosquito bites. Irradiated mosquitos were allowed to feed on volunteers for about 20mn and each blood-engorged mosquito was then dissected to determine whether its salivary glands contained sporozoites. Control volun- teers were bitten by a comparable nuimber of non- infected irradiated mosquitos. In addition, one or more volunteers were usually exposed to a small number of nonirradiated infected mosquitos in order to verify the infectivity of irradiated mosquito batches. The immunization procedure was repeated with the same volunteers 2-8 times at intervals of 2 or more weeks. Following completion of their immunization sched- ules, volunteers were challenged with nonirradiated infected mosquitos approximately 2 weeks after their last exposure to irradiated mosquitos. On each occasion, at least one conti ol volunteer was also bitten by the same mosquitos in order to verify trans- mission potential. Mosquitos were divided into 2 262 IMMUNIZATION WITH ATTENUATED SPOROZOITES groups and allowed to feed intermittently on immu- nized and control volunteers by the interrupted bite technique (4). After feeding, mosquitos were dis- sected to determine the actual number tbat were infected. The protective effects of immunization were deter- mined by examination of thick blood films obtained from control and immunized volunteers. Blood films were obtained 7 days after challenge and, depending on the development of patent parasitaemia, daily for the next 3 weeks, every second day for the following 2 weeks, and twice a week for another 4. months. Im- munized volunteers who failed to develop parasit- aemia within several weeks after challenge were ex- posed again to mosquitos infected with a homologous or heterologous strain of P.falciparum. Volunteers who developed an acute clinical attack of malaria were given antimalarial treatment to cure their infec- tions. As a precautionary measure, radical curative treatment was also administered to participants who did not develop patent parasitaemia during the study. Participants who were exposed to P. vivax sporozoites received the standard chloroquine-primaquine treat- ment and those exposed to P.falciparnm sporozoites were cured with chloroquine (exposure to Tamenie strain) or amodiaquine-tetracycline (exposure to Marks strain). RESULTS Immunization against P. vivax Three persons who were exposed to irradiated mos- quitos infected with P. vivax were not protected against subsequent challenge with the homologous strain. They were bitten by irradiated mosquitos on 4 occasions at intervals of 2-4 weeks. The prepatent period, development of parasitaemia. and clinical course of infection in these volunteers were similar to those observed in control volunteers. Immunization against P. falciparum Exposure to fewer than 200 irradiated infected mosquitos on 2-4 occasions. Four volunteers who were exposed on 2-4 occasions to irradiated mos- quitos infected with the Tamenie strain of P.falci- parun were not protected against subsequent challenge with the homologous strain. They were each bitten by a total of fewer than 200 irradiated infected mosquitos over a period of 1-4 months. During the course of immunization, 2 of the volunteers devel- oped patent infections, which were cured immediately with chloroquine. The course of infection in these individuals after challenge was similar in every respect to that observed in control volunteers. Exposure ro 440 or more irradiated infecred mos- quitos on 6-8 occasions. Three volunteers-L.A., D.S. and W.D.-were immunized over a period of 10-38 weeks by allowing a total of440-987 irradiated mosquitos infected with the Tamenie strain of P.fal- ciparum to bite them on 6-8 occasions (Fig. 1). TOTAL MOSQUITOES. 50 40 45 65 20 220 440 VoI.iler LA 20 220 140 77 QS Vollo 0|erDS 140 77 Vduni5tr WD ;200 96 135 56 954 500 96 546 I i- - - -- 6 6 I& 36 32 28 24 20 16 12 a woo-II 17 9"7 4 0 FINAL + IMMUPI LATION Fig. 1. Immunization schedule of 3 protected volunteers. Ir- radiated mosquitos infected with the Tamenie strain of Plas- modium fa1ciparum from Ethiopia were allowed to introduce the sporozoite antigen by feeding Numbers above the arrows ind;cate the number of infected mosquitos feeding on the volunteer. Volunteer L.A. was exposed to irradiated mosquitos on 6 different ocCasions and the interval between exposures was precisely 2 weeks on each occasion. Dissection of blood-fed mosquitos over the 10-week period revealed that he had been bitten by a total of 440 mosquitos infected wich the sporozoites. Two weeks after the last immunizing exposure, L.A. was challenged with 13 nonirradiated mosquitos infecred with the same strain of P.falciparum. He did not become infected, whereas the conTrol volunteer de- veloped patent parasitaemia 12 days later. However, after being challenged again with 45 Infected mos- quitos. 16 weeks after the last immunizing dose, L.A. developed an infection during the normal prepatent period (Table 1). Volunteer D.S. was exposed to irradiated mosquitos on 8 different occasions and was bitten by a total of 954 irradiated mosquitos containing sporozoites. The first 4 immunizing exposures were given at regular intervals evenr 2 weeks. There was then an interval of about 13 weeks between the fourth and fifth ex- posures. A period of 6 weeks elapsed before the next exposure, and the last 2 exposures were given once more at intervals of 2 weeks. The course of immu- nization extended over a period of 30 weeks. Two weeks after the last immunizing dose, D.S. was bitten by 14 nonirradiated mosquitos infected with the s .1.63 K. H. RIECKMANN ET AtL. Table 1. Challenge of immunized volunteersa with sporozoites of two strains of Plasmodium falciparum Wees fotowing final vaoccnation Volunteer L A Volunteer D S Volunteer W D No of No of No of Strain nosquitos Patency Strain mosquitos Patency Strain rnosqunos Patency 2 Tarnen,eb 13 No Tamenie 14 No 4 6 B Tameuiie 12 No Mearks c 9 No 10 12 14 16 Tamenie 45 Yes 18 Marks 7 Y35 Trnenie a Yes B Al control volunteers became positive within 14 days after being bitten by the sarne mosquitos used to challenge imrnunized volunteers b The Tamen. strain ongrnated in Ethop.a C The Marks strain onginated in Viea Nam homologous Tameme strain. He did not develop a patent infection. Approximately 8 weeks after the last immunizing dose, he was rechallenged by 12 mos- quitos infected with the same homologous strain and, again, he was fully protected. On both occasions, control volunteers developed patent infections within 2 weeks after challenge. The immunized volunteer was not protected after challenge by 7 mosquitos in- fected with the heterologous Marks strain of P.fakciparum 17 weeks after the last immunization (Table 1). He was also not protected against challenge with the homologous Tamenie strain 25 weeks after the last immunization. * Volunteer W.D. was exposed to irradiated mos- quitos on 7 different occasions and was bitten at irregular intervals over a period of 38 weeks by a total of 987 irradiated mosquitos containing sporozoites. About 8 weeks after the last immunizing dose, he was challenged with 9 mosquitos infected with the Marks strain and was protected against this heterologous challenge, whereas 2 control volunteers became patent within 11-13 days after challenge. Eighteen weeks after his last immunizing dose, the immunized volun- teer was challenged again by mosquitos infected with the homologous Tamenie strain, and developed patent parasitaemia 13 days later (Table 1). DISCUSSION The results of the investigations described in this report indicate that protection against malaria can be induced in man by inoculation of irradiated sporo- zoites of P.falciparum. Considered together with findings reported earlier (5-8), 5 persons have been successfully vaccinated against falciparum malaria. Under our experimental conditions, protection was achieved only after 6, 7, or 8 exposures to a total of 440-987 irradiated infected mosquitos. Exposure to a lotal of fewer than 200 irradiated infected mosquitos on 2-4 occasions failedto protect 4 individuals against P.Jalciparum and 3 individuals against P. vivax. Indi- viduals who were protected did not develop patent parasitaemia, whereas those who were not protected developed patent infections; these infections were indistinguishable ftom those observed in nonimmu- nized persons. The results agree with observations in animal models that immunity induced tco sporozoites does not extend to the blood stages of the malaria parasite (9, 10). Exposure to irradiated infected mosquitos could not be carried out at well-defined intervals or with a constant number of infected mosquitos since immu- nization depended on the availability of gametocyte donors to infect the mosquitos. Difficulty in obtaining gametocyte donors was due to variability in the res- ponse of infected individuals to suppressive drug regimens, in the duration and intensity of subsequent gametocyte levels, and in the infectivity of gameto- cytes to mosquitos. These difficulties, considered together with the problem of estimating the number of sporozoites released during mosquito feeds, pre- clude any definite estimates of sporozoite inocula or optimum immunization schedules at the present time. During the course of immunization, patent para- sitaemia developed in 2 of the 7 persons who were 264 IMMUNIZATION WITH ATTENUATED SPOROZOITES 265 bitten by fewer than 200 mosquitos. These obser- vations confirm those made by Clyde et al. (6) that irradiation with 120 Gy is not always effective in ren- dering all sporozoites noninfective to man. Further studies are needed to determine the minimum amount of irradiation that is required to ensure that no sporo- zoites remain infective. Protection was observed for at Jeast 8 weeks follow- ing 6-8 exposures, but it was no longer evident 16-18 weeks after immunization. Of particular importance is the fact that this protection extended to a strain of P.falciparun geographically remote from the strain used during the course ofimmunization. These obser- vations suggest that antigenic variation, observed among asexual erythrocytic stages (11), may not inter- fere significantly with the induction of immunity by sporozoites. The results obtained in these studies are essentially in agreementwith those described by Clyde et al. (5, 6) and they provide substantial evidence that attenuated sporozoites can protect people against falciparum malaria. In viewl of current technological constraints, it seems unlikely that a sufficient number of sporozoites would be forthcoming to immunize large population groups. On the other hand, a sporozoite vaccine might be produced for administration to small groups of nonimmune individiuals in areas of high malaria endemicity. With the availability of an effective vaccine, such individuals would no longer have to be concerned about taking prophylactic dmgs at regular intervals or becoming infected with drug- resistant strains of Pfalciparun. The successful immunization of human volunteers reported here, and by others, should encourage continuing efforts toward the development of a sporozoite vaccine against human malaria. RESUME EMPLOI DE SPQROZOITES ATTfENUS DE PLASMODIUM FALCIPARUAf POUR L IMfMUNISATTION DE VOLONTAIRES HUMAMNS Apres leur Inmmunisation au moyen de sporozoites att6nu6s de la souche ethiopienne Tamenie de Pkasnrodium fakciparum, on a constate chez trois volontaires humains une protection efficace contrc les sporozo,tes normaux. Les spo- rozoltes ayant op6r8 l'imnminisation avaient ete att6nu6s en soumettant les moustiques porteurs (Anopheles sephens:) A une dose de rayonnements ionisants d'au mains 120 Gy (12 000 rad). Les moustiques infect6s et irradies ont pu ensuite se nourrir sur les volontaires et injecter ainsi dans Ieur sang des sporozoites. Pendant les 10 i 38 semaines qu'a pris ce processus d'immunmsation et k intervaIle de temps variable, des volontaires ont 6t6 exposes 6 i 8 fois aux piqflres de 440 i 987 moustiques infectes et irradiis au total. La protection contre une infection ut6rieure par des mous- tiques non irradiEs a dur6 au moins 8 semaines apres la der- nikre "injection" de sporozoftes irradiks mais elle avait pris fin lors d'epreuves faites les 16eme ou J86me semaines. Ceux des volontaires qui n'avaient e exposes qu'A un nombre de moustiques 6gal ou inferieur A 200 en 2 a 4 fois n'ont pas, en revaanche, et6 immunises contrc l'infection par des mous- tiques non irradies. L'immunisation oar les piq&res d'un nombre suffLsant de mnoustiques irradi6s porteurs du para- site de la souche ethiopienne Tam6nie sensible a la chloro- quine s'est r&v6I6e egalement capable de protiger contre la souche vieEnamienne Mlarks de P.falciparum resistant A la ctllorocuine. Ces resultats donnent i penser que l'immuni- sation au moyen de sporozoites at:enues peut constituer une methode utile pour proteger de petits groupes d'individus non immuns vivant dans des zones d'cndemie. Its justifient Ia poursuire des efforts visant A mettre au point un vaccin A base de sporozoltes concre le paludisme hurnain. REFERENCES 1. NuSSENZWE[G, R. S. ET AL Nature (London), 216: 160-162 (1967). 2. RJECKMANN, K. H. ET AL. Proceedings of the Helmin- Ihological Society of Washington, 39 (Special issue): 339-347 (1972). 3. EARLE, W. C. & PEREZ, MI Journal oflaboratory and clinical medicine, 17: 1124-1130 (L932). 4. COATNEi, G. R. ET XL. Amnerican joujrnial of hygiene, 47: 113-119 (1948). 5. CLYDE, D. F. ET AL American journal of the medical sciences, 266: 169-177 (1973). 6. CLYDE, D. F. ETAL. American journal of the nmedical sciences, 266: 398-403 (1973). 7. RIECKMANN, K. H. ET AL Transactions of the Royal Society of Tropica Medicine and Hygiene. 68: 258-259 (1974). 8. CLYDE, D. F. ET AL. American journal of tropical medicine and hygiene, 24: 397-401 (1975). 9. NLSSENZWEIC. R. S. ET AL Nature (London), 222: 488-489 (1969). 10. BEAUDOIN, R. L. ET AL. 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