Recent developments in vaccination against malaria: Immunization of experimental monkeys against Plasmodium falciparum: use of synthetic adjuvants*

Budlermn of t/e World Health Organizaton, 57 (Suppl. 1) ' 199-203 (1979) Immunization of experimental monkeys against Plasmodium falciparum: use of synthetic adjuvants* W. A. SwDDlQU1, DIANE W. TAYLOR, 2 S. C. KAN,2 K. KRAMER,' S. M. RICHMOND-CRUM,3 S. KOTANI,4 T. SHIBA,5 & S. KASUMOTOS The replacement of Freund's adjuvant by a possible safe adjuvant for effective im- munization of owl monkeys (Aotus trivirgatus gnseimembra) against a huwnan mnalaria parasite, Plasmodium falciparum, has been investigated. Experiments involved the use of two synthetic adjuvants: MDP (N-acetylmruramyl-L-alanyl-D-isoglutamine) and stearoyl- MDP (6-O-stearoyl-N-acetylmuramyl-L-alanyl-D-isoglutamine). In both cases, P. falciparum merozoites obtained through short-term in vitro cultivation were used as antigen. MPD wvas used as adjuvant in 5 owl monkeys; 2 control tnonkeys died and of the 3 experimental monkeys only I survived. In contrast, in another experiment where stearoyl-lMDP was used as adjuvant, there was I00% protection of4 inmmunized monkeys against a challenge with the homologous strain ofP. falciparum. The results of thesecond experiment are encourag- ingfor the developmnent ofan effective andsafe vaccinefor hunan malaria. Attempts to find a vaccine against malaria began half a century ago but gave way to searches for new drugs during thXe Second World War and to anti- mosquito programmes during the post-war years. However, resistance to drugs and insecticides and lack of money have reduced the prospects of malaria eradication and thoughts have again turned to the possibility of developing a malaria vaccine (1). Four different kinds of*vaccines are currentlyunder investigation: exoerythrocytic merozoites raised in tissue culture (2), irradiated sporozoites from the mosquito (3-5), extracts from blood schizonts (6-9), and emulsified erythrocytic merozoites (10, 1)). Most of the above studies have been made on bird, rodent, and monkey malarias. This report deals with studies made on a human malaria parasite, Plasmodium faciparum. A year ago a report was published from this labora- tory (12) showing the first successful immunization of * Tlus work was supported by Lhc Agency for International De- velopment, US Department of State, Contract No AJD/ta-C-1227. Professor, Department of Tropical Medicine and Medical Microbiology, University of Hawaii School of Mcdicine, Honolulu, HI 96816, USA. - Scnior Research Associate, Department of Tropical Medicine andMedicaL Microbiology, University of Hawami School of Medicine. Honolulu, HI 96816, USA. I Rcsearch Associate, Department of Tropical Medicine and Medical Microbiology, Umversity of Hawaii School of Medicine, Honolulu, HI 96816, USA. I Professor, Department of Microbiology, Osaka University Dental School, Osaka, Japan. I Professor, Faculty of Science, Osaka University, Toyonaka, Osaka, Japan Aotus trivirgatus griseimembra monkeys against P.falciparum infection. This result was rapidly con- firmed (13). In these studies the use of Freund's complete adjuvant was found to be essential for effec- tive immunization. The use of Freund's adjuvant in man is not considered safe because serious side-effects have been widely reported: potentiation of plasma cell tumours in mice, induction of autoimmune reactions, formation of dissemninated focal granulo- mata, and long-term persistence of mineral oil in animals (14-18). The development of an immuno- logically satisfactory and pharmacologically accept- able adjuvant is of prime importance to the de- velopment of an acceptable human malaria vaccine. The synthesis ofN-acetylmuramyl-L-alanyl-D-isoglu- tamine (muramyl dipeptide, MDP) has been achieved recently in two laboratories (19, 20) and it has been shown to be capable of replacing the whole tubercIe bacilli and of enhancing the immune response of an animal against an antigenwhen injected with Freund's incomplete adjuvant. However, before this compound could be used for imunuization in man it would be necessary to eliminate the mineral oil, which is partly responsible for undesirable side-reactions. A recent report from one of our laboratories (21) has shown that the replacement of the primary hydroxyl group at the C-6 position of MDP by a lauroyl, stearoyl, or docosanoyl group gave an MDP derivative with adju- vant activities. This report describes the results of immunization experiments inAotus tr:virgatusgrisei- membra with P.falciparum antigen using MDP and modified MDP as adjuvants. 3385 -199- 200 W. A. SIDDJQUl ET AL. MATERIALS AND METHODS Preparation ofantigen The Uganda-Palo Alto strain (FUP) of P.falci- parum used in this study is maintained in the labora- tory by serial passages of blood-induced infections in owl monkeys (22) and by continuous in vitro culture (23). The antigen was prepared by short-term in vitro cultivation of theFUP strain ofP.faciparum derived from infected owl monkeys. RPMI 1640 medium sup- plemented with fetal calf serum (FCS) and fatty acid- free bovine albumin (FAF albumin) was used for short-term in vitro cultivation ofP.falciparum. Para- sitized blood from owl monkeys was cultured in sterile 500-mi side-arm flasks fitted with stoppers with entry ports for a gas mixture containing 90/o N2, 8w/s COC, and 2%o 02. Seven millilitres of heparinized, para- sitized blood, washed twice with medium, were intro- duced into each culture flask containing 63 ml of RPMI 1640, 8.8 ml ofFCS, and 6 ml ofFAF albumin (12.5 g/litre). The medium was changed at approxi- mately 12 and 24 hours of incubation. At the end of 35-40 hours' incubation, most of the parasites had developed to mature segmenters containing fully de- veloped individual merozoites. These mature seg- menters were concentrated and harvested relatively free of other cellular elements as described previously (24). In the final preparation, as described in an earlier report (12), the antigenic material consisted of 50-60'o segmenters containing individual merozoites and the remainder consisted of other developmental stages of the parasite. The antigen was stored at -20 "C. Adjuvants andpreparation of vaccine MDP-peanut (groundnut) oil. MDP was syn- thesized im one of our laboratories (20). 1.5 ml of Arlacel A° was added to 8.5 ml of peanut oil. The mixture was autoclaved and stored at 4°C. Three hours before vaccinating the experimental animals, 0.5 ml of the parasite antigen was mixed thoroughly with 0.5 ml of the peanut oil carrier containing 125 Mg of freshly added MDP. Details of the composition of the vaccine and the vaccination schedule are given in Table 1. Stearoyl-MDP and liposomes. 6-0-stearoyl-N- acetylmuramyl-L-alanyl-D-isoglutamine was syn- thesized in one of our laboratories (21). The adjuvant- incorporated liposomes were prepared following the description of Inoue (25) with some modifications: 10 Jmol of cholesterol (Grade 99 + /o)' and 10 umol of lecithin (dipalmitoyl-DL-phosphatidyl choline, Grade I approximately 99%oa were dissolved in 5 ml a From the Sigma Chemical Co., SE Louis, MO, USA. Table 1. Summary of vaccination studies in owl monkeys against Plasmodium falciparum using MDP as an adjuvant Pro- Peak Aotus patent pra- monkey perod stawnin Death or No Cornpositlon of vaocre (days} (tEd surva A232 Control (lne) 3 60 6 did on dly 14 A231 Control (MDP + peonut I) 3 48 8 ded on day 14 A2D00 Ag + MOP + peanutol 7 90 4 ded on dey 13 A201 Ag + MDP + pemnutolC 11 84 survied A222 Ag + MDP ppenutodc 10 444 diedon day26 a A2, A231, endAS wer challenged with 2 6 x 10' FUP paraetes whie A200 and A201 were gen 5 x 10' FUP parvaste b 125 jwg of MDP In 0 SrnJ of peanut oi given intramuscularly on dlysO. 12, 33, and 69, L FUP-werozot-nrlchedantigen eulatd with125Igaf MDP in0.5mlof peanut o gien intramuscularly on deys 0, 12, 33, and 69. of chloroform in a 10-ml round-bottomed flask. All the chloroform was removed by means of a rotary vacuum evaporator below 30 °C. The adjuvant (0.025, 0.5, or 1.0 mg) in 0.5 ml of phosphate-buf- fered saline was added to the lipid-coated flask. Lipo. somes were then prepared by sonication at 0 IC under argon. The vaccine was prepared by thoroughly mixing the adjuvant-incorporated liposomes with antigen, using a double-hubbed needle and two syringes. Further details of the composition of the vaccine and the vaccination schedule are given in Table 2. RESULTS AND DISCUSSION Immunization studies usingMDP as an adjuvant Five monkeys (Aotus trivirgatus griseimembra) weighing approximately 800 g each were used in this experiment (Table 1). Two monkeys (No. A232 and A231) were used as controls; the other 3 (No. A200, A201, and A222) were immunized with P.falciparum (FUP strain) merozoites emulsified with an equal volume of purified peanut oil containing 125 ug of MDP. This vaccine was administered intramuscularly 4 times to each of the experimental monkeys, a total of 2.88 mg of antigen protein being given to each animal. Immunization never produced a detectable infection. On day 111, 6 weeks after the fourth vac- cination, all 5 monkeys were challenged intravenously, with 1 x 103 parasites (FUP strain of P.falciparum). None of the 5 animals developed a patent parasit- aemia, as monitored by daily thick filmns. On day 31, 4 weeks after the first challenge inoculum, all animals were again challenged with 5 x 104 PUP parasites. Seven days after the second challenge, No. A200 became patent and died 6 days later with a >90°o parasitaemnia. Monkey A201 became patent on day ADJUVN-TS FOR INIMUNIZATION Table 2. Immunization of owl monkeys against Plasmodium falciparum (Uganda- Palo Alto (FUP) strain) malaria Compositon of vaccinea Parasite protein Paraste orotein Number of infected KGS b pep Adjuvant C (mg) in 0.5 nt of (mg) in 0 5 ml of Total erythrocyes in the injection plus liposome d KGS (injecton 1. KGS (injection 2. oaresite protein challenge noculum Monkey No Iml) (ml) ddy Olc day 28) e (mg) (day 451 f A343 7.5x10' A291 15 75x10' A 287 0.5 1 0 - - - 7 5 x 105 A 286 - 1 0 1 0 1 86 2.86 7 5 x 10 A 284 - 1 0 1 0 1.86 2 86 7.5 x 105 A23 - 1.0 1 0 1 86 286 75x 10' A294 - 1 0 14 14 2.8 75x 10' Adiuvant wth liposoml plUs antigen mxed, using a double-hubbed needle and two syringes, adninistered intramuscularly in alternate thighs b Kreb's glucos aline C Synthetc 6-O-steeroyl-N-acetylmuramyl-L-alanyl-D-soutamine A total of 1.5 mg of this adjuvant was given to each of the monkeys A 287, A 288. A 284. and A 283, wsle onty 0.5 mg was given to A 294 d Liposon-s contained lecilhin and cholesterol and was prepared followng the rnothod of Inoue t25) More than 50% aegmenlers contairng indvidual merozoltes Tne remainder o1 the parasite mnatenal consisted of other developmental stages of the parasite t Parastized blood was obtained from an infected (FUP) owl rmonkey. The inoculumn was given introvenouslv. 11, developed a peak parasitaemia of 8.407o on day 23, and cleared the infection by day 29, after the second challenge. All other monkeys remained negative. On day 90, 3 months after the first challenge. all surviving monkeys were challenged with 2 x 10Q FUP parasites. Both control monkeys were patent on day 3 and died on day 14-No. A232 with a 60.67o parasitaemia and No. A231 with a 48.801o parasitaemia. Monkey A222 became patent on day 10 and succumbed to the infec- tion on day26 with a 200o parasitaemia. No. A201 de- veloped a very low-grade infection, with less than 0.1/o parasitaemia for 9 days, becoming patent on day 4 and clearing the infection by day 12, after the third challenge. The results of this experiment are similar to, but somewhat better than, those obtained by Voller & Richards (26), who failed to protect owl monkeys with Freund's complete adjuvant and formalin- treated infected cells. In our present experiments, monkeys immunized with P.falciparurn merozoite antigen mixed with MDP-peanut oil showed a de- layed onset of parasitaemia, with one monkey (A201) surviving. Attempts to establish the minimum number of parasites needed to infect an owl monkey resulted in a number of unsuccessful challenges. However, these challenges did not alter the course of infection in the controls and should not detract from the survival of monkey A201. Immunization studies using stearoyl-MJDP and liposomes Seven owl monkeys weighing approximately 900 g each were used in this experiment. The physical characteristics and coloration of these monkeys indi- cated that they were of phenoty-pe group B, according to the description ofMa et al. (27). Using the standard chromosome preparation method of Moorehead etal. (28), these monkeys were found to belong to karyo- type II as defined by Ma et al. (27). The composition of the vaccine and the immunization schedule are summarized in Table 2. Three monkeys (No. A287, A291, and A303) were used as controls; the other 4 monkeys (No. A283, A284, A286, and A294) were immunized with P.fakc:panum (FUP strain) mero- zoite-enriched antigen mixed with the adjuvant- incorporated liposomes. This material was adminis- tered intramuscularly on two occasions, 4 weeks apart, to each of the 4 experimental monkeys. A total of 2.86 mg of parasite protein (1.0 mg on day 0 and 1.86 mg on day 28) was administered to 3 monkeys (A286, A284, and A283). Mlonkey No. 294 received a total of 2.80 mg of parasite protein (1.4 mg on day 0 and on day 28). Immunization never produced a de- tectable infection. On day 45, that is, 17 days after the second inoculation, all 7 mnonkeys were challenged by intravenous injection of 7.5 x 105 parasites (FUP strain of P.fatciparum) derived from an ongoing in- fection in an owl monkey. Thick or thin blood films were made daily to follow the course of infection in all monkeys and the results are summarized in Fig. 1. The two control monkeys (A303 and A287) died within 1-2 weeks after the chal- lenge. Monkey No. 303 died on day 8 with a 60.0%o infection and No. A287 died on day 15 with 54.4% in- .)01 W. A. SIDDIQUI ET AL. U -- , Li 0 40i AZ3I(A AZIO I~~ ~ ~ ~ ~ cAll? _n J i A?"4ELI3 IX :R IIJ 3C t^VALCMATED ( 234 20- 10- ~~~Id *t 'P+ *\*._ 0 5 .0 15 20 25 30 3 DAYS AlTER CHALLENIGE Fig. 1. Course of infection of Plasmod,um falciparum (FUP strain) in control and vaccinated monkeys (Aotus trivirgatus griseimembra). fection. Although A291 eventually survived the infec- tion, it attained the peak parasitaemia of more than 25%. In contrast, all 4 immunized monkeys survived. In immunized monkeys A286 and A294, low-grade infections lasted for I week, while in A284 and A283 the parasitaemia ranged between 5.0 and 14.0% for 1 week. By day 24, A284, A283, and A294 had become negative and no parasites could be detected in A286 by day 32. Up to 6 months after the challenge, all these monkeys remained negative. Although the number of monkeys used in this experiment was small, the difference between the course of infection in immunized and nonimmunized monkeys is indeed very significant. Spontaneous recovery of a control monkey (A291) is a very rare occurrence. Within the last 7 months, blood-induced infections (FUP strain ofP.falciparum) were initiated in the same manner in 3 other Aotus trivirgatus griseimembra (A306, A290, and A275) and all the 3 monkeys showed a typical course ofinfection and died (Fig. 2). These 3 monkeys were also of karyotype I1 and phenotype group B. It is concluded from these results that P.falciparum merozoite immunization, using 6-0-stearoyl-N- acetylmuramyl-L-analyl-D-isoglutamine with lipo- somes as an adjuvant, protects against homologous infection with intraerythrocytic stages of the normally lethal P.fakciparum parasites. The administration of P.falciparum antigen with adjuvant and liposomes did not produce any reaction at the site of injection. The only side-effect associated with the adjuvant was anorexia for a few days immediately after vaccination, which resulted in some-loss of weight. However, all immunized and adjuvant control monkeys regained weight within 2 weeks following immunization. The loss of weight was considerably less in A294, which re- ceived only one-third the concentration of the adju- vant given to the other monkeys. This is an important observation, indicating some correlation between the concentration of adjuvant used and the degree of side-effect observed. Experiments are in progress to define the optimal concentration of this new adjuvant that is efficacious and has minimal side-effects. There are 6 reports to date on studies in owl monkeys of immunization against P.fatciparum. In two studies, the use of P.falciparum ring stages as antigen failed to afford complete protection of the immunized owl monkeys (26,28). In contrast, in two other studies (12, 13) in which P.fazciparum mero- zoites emulsified with Freund's complete adjuvant were used as vaccine, complete protection of the im- munized monkeys was obtained. The replacement of Freund's complete adjuvant by MDP did not give 100% protection in two other studies (23; W. Trager et al., personal communication). However, the exper- iments reported above show the first successful replacement of Freund's complete adjuvant by 6-0- stearoyl-N-acetylmuramyl-L-analyl-D-isoglutamine for effective immunization of owl monkeys (Aolus trivirgatus griseimembra) against infection with a human malaria parasite, P.falciparum. These resuts are encouraging and provide good grounds for believ- ing that an effective and safe vaccine for human malaria may be developed in the not too distant future. 701 b 0 0 En0. D,.e o AvZ? * A306 * A290 0 Fig. 2. Course of infection of Plasmnodium falciparum (FUP strain) in normal Aotus trivirgatus griseimembra monkeys. 202 ADJUVANTS FOR IMMUNIZATION 203 RtSUMt INIMUNISATION DE SINGES D'EXPtRIENCE CONTRE PLASMQDIUMFALCIPARUA.t EMPLOI D'ADJUVANTS SYNTHETIQUES Pour 6tudier la possibiLlk de substituer a I'adjuvant de Freund un adjuvant pr6sentant moins de risques d'effets secondaires. on a 6valu6 V'efficacit6 de la vaccination de singes nocturnes (A otus trivirgatits gruserrnenibra) contre urn parasite infectant I'bomrne, Plasmodium faciparwun. Les exp8riences ont Wt6 faites avec deux adjuvants synthitiques diff6rents: MDP (N-acety]-muramyl-L-alanyl-n-isogluta- mine) et st6aroyl-MDP (6-O-stearoyl-NV-acetvl-muramy1-L- alanyl-D-isoglutamine). On a utilise dans Ies deux cas comme antig6ne les mErozottes de P.falkrparum obtenus au moyen d'une culture in vitro de courte duree. Lexperi-nce avec MDP a porte sur 5 singes nocturnes: les 2 singes servant de contr6les sont morts et un seul des 3 singes soumis i L'epreuve a surv6cu aux inoculations ult6rieures de parasite. Par conEre, dans 1'expernence utnlisant stearoyl-MDP comme adjuvant, une protection A 100% a &ik constat6e chez les 4 singes vaccLn6s Iorsqug ceux-ci ont et6 exposes A une souche deP.falciparum homologue. Quant aux 3 singes servant de controles, ils ont contract6 la maladie ct 2 d'entre eixx y ont succomb&. Les r6sultats de la seconde exp6rience permettent d'envisager avec optimisme les perspectives de mise au point d'un vaccin contre le paludisme humain qui soit A la fois efficace et satisfaisant sur lc plan de la s6cuntL. REFERENCES 1. WHO Technical Report Series, No. 579, 1975 (De- velopments in malaria immunology; repert of a WHO Scientific Group). 2. HOLBROOK, T. W. ET AL. Journal of parasitology, 60: 348-354 (1974). 3. NUSSENZWEIG, R. A. 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