Recent developments in vaccination against malaria: Nor-MDP, saponin, corynebacteria, and pertussis organisms as immunological adjuvants in experimental malaria vaccination of macaques

Bulletin of the World Health Organization, 57 (Suppl J) 189-197 (1979) Nor-MDP, sapomnn, corynebacteria, and pertussis organisms as immunological adjuvants in experimental malaria vaccination of macaques G. H. MITCHELL,' W. H. G. RICHARDS,2 A. VOLLER, F. M. DIETRICH,4 & P. DUKOR' Vaccination ofprimates against malaria using antigen derivedfrom erythrocyticpara- site stages has been most successful where Freund's complete adjuvant has been employed. Since this adjuvant is clinically unacceptable its replacement is a matter of urgency. In the present work a nmuramyldipeptide derivative (nor-MDP) given in mfneral oil has proved to be partially effective as an adjuvantfor merozoite vaccination ofMacaca mu]atta against Plasmodium knowlesi, andsaponin hasproved to be effective in similar vaccination of M. fascicularis. Merozoite vaccination using Freund's complete adjuvant (FCA) has proved effective in protecting Macaca mulatta against challenge with Plasniodium knowlesi and Aotus trivirgatus against challenge with P.fakiparum (1). The extreme reactivity of FCA pre- cludes its consideration as a potential clinical adju- vant in malaria vaccination (2); hence it is imperative to find an effective replacement. The present experi- ments involved preliminary investigation of several other substances and organisms as adjuvants for P. knowlesi merozoite vaccine in two macaque species, M. mulatta and M.fascicularis.. MATERIALS AND METHODS Monkeys Indian-born rhesus monkeys (Macaca mulatta mulatta) and a Malaysian-born kra monkey (Macaca fascicularis) were obtained as imported, conditioned juveniles and young adults, weighing between 2 and 3.5 kg. The imported kra was treated with anti- malarials and splenectornized before use, as pre- Research Assistant, Department of Chemical Pathology, Guy's Hospital Medical School, London SE] 9RT, England. ! Principal Scientist, Protozooiogy Departmer.t, Wvellcome Laboratones of Tropical Miedicine, Langlev Court, Beckenham, Kent BR3 3BS, England. Porrit Senior Lecturer. Department of Clnical Tropical Med- cine, London School of Hygiene andTropical hedicine, arid Nuffield Laboratory ofComparauve Medicine, Institute of Zoology, Regent s Park, London NW1 4RY, England. 4Staff Member, Immunology Section, Research Laboratories, Pharmaceuttcals Division, CIBA-GEIGY Ltd.. CH 4002 Basle, Switzerland. I Head, Immunology Section, Research Laboratories, Pharma- ceuticals Division, CIBA-GEIGY Ltd . CH 4002 Basie, Switzerland. viously described (3). Captive-born kra monkeys had been bred in the United Kingdom and were used as adults weighing 3-7 kg. All the monkeys were caged in rooms artificially illuminated for 12 h per day. Water and Zoofood Old World monkey dieta were continuously available and fresh fruit and green veg- etables were fed frequently. Parasites Plasmodium knowlesi parasites of two established laboratory strains designated Washington (4) and Nuri (5) were used in these experiments. Both were isolated in Malaysia and cause fatal fulminant malaria in almost all normal rhesus monkeys infected by intra- venous inoculation. Parasites were maintained as frozen stabilates in 150 g/litre (lI5 Vo) glycerol saline at -70 °C or by passage between normal monkeys. Distinct intra-strain variants ofWashington(W) strain parasites were identified by the schizont-infected red cell agglutination (SICA) test (6). Antigen Throughout these experiments all animals were vaccinated using WI variant merozoites. Merozoites for vaccine antigen were cultured and isolated as pre- 'siousJy described (7, 8) from short-term schizont cultures, treated with phytohaemagglutinin, and gently centrifuged after substantial nerozoite release to leave merozoite-rich supernatants depleted of blood cells. Supernatant samples were counted in haemocytometer chambers, and the suspensions cen- trifuged (3000 g, 10 min) to pellet the merozoites. c Fromr. BP Nutrition Ltd. WXVham, England 3837 -189- 190 G. H. MITCHELL ET AL. After discarding the cell-free supernatant, the pellets were rapidly resuspended in sufflcient cold, sterile 10% normal rhesus serumnin distilledwater to produce a concentration of 109 merozoites per millilitre. This suspension was stirred magnetically on ice while 1-mil aliquots were dispensed rapidly into 6-ml capacity sterile, round-bottomed glass ampoules, which met the criteria of Campbell (9). Ampoules were plugged with cotton wool and plunged into liquid nitrogen until freeze-drying commenced; alternatively, they were warmed to -20 IC, sealed, and returned to liquid nitrogen for storage until use. Freeze-drying (10 h, -35 IC, at a pressure of 13-26 Pa), secondary desiccation (170 h over P20; initial pressure 1.3-2.6 Pa), illing with dry nitrogen gas, and sealing of the ampoules were carried out by the Standards Processing Section, National Institute for Biological Standards and Control, according to the protocol of Campbell (10). The ampoules were stored at -20 °C for up to 26 weeks before use. Rhesus monkeys of groups A and B (Table 1) re- ceived vaccine processed by one of the authors (G. H. Mitchell) by freeze-drying for 18 h and sealing under reduced pressure (5.3 Pa) without secondary desiccation. This was stored at4°C for up to 20 weeks before use. Adjuvants usedfor vaccination of rhesus monkeys A muramyldipeptide derivative nor-MDP (N- acetylglucosamine-3-yl-acetyl-L-alanyl-D-isoglu- tamine) was synthesized by Dr J. Stanek and Dr A. Hartmann (CIBA-GEIGY, Basle). 5 mg of nor-MDP dissolved in 1 ml of phosphate-buffered saline and mixed with I ml of Freund's incomplete adjuvant (FIA) was used for a single adjuvant dose. Mfycobacterium butyricum was obtained as kiUed and dried cultured bacteria, Bacto batch No. 634804, or in suspension in light mineral oil with Arlacel A emulsifier as Freund's complete adjuvant (FCA).b 0.5 mg of M. butyricum was used for an adjuvant dose. Freund's incomplete adjuvant (FRA)c consisted of light mineral oil and the emulsifier only. Adjuvant 65d consisted of I g of alumninium monostearate and 5 g of mannide monooleate per 53 g ofgroundnut (Arachis hypogaea) oil, sterilized at 160°C for 1 h. Oil emulsion adjuvants consisted of I ml of oil phase and I ml of aqueous phase per dose. A suspension of Bordetella perfusis organisms, 8 x 1010 per ml, was kindly provided by Wellcome b Batch no. 641896 from Difco Laboratonies, Detroit, Ml, USA. c Batch no. 592109 from Difco Laboratories, Detroit, MI, USA. d Prepared by us according to Patent Ncth. 6,402,687 (Merck & Co. Inc.). Research Laboratories, Beckenham, England. One human vaccinating dose (2 x 1010 organisms) was used for each adjuvant dose. A djuvants usedfor vaccination ofkra monkeys Freund's complete adjuvant and B.pertumssis were used as specified above. Corynebacterium parvum suspension (Coparvax, Wellcome Research Labora- tories, Beckenham) contained 7 mg of coryneforms per ml. I mg (approximately 2 x 109 organisms) was used as an adjuvant dose. Saponin SPL (a former product of Boake Roberts Ltd.; no longer available) was a preparation from the ground bark of Quillaja saponaria. I mg was used as an adjuvant dose. Preparation and administration of vaccines Merozoite antigen (10' merozoites per dose) was thawed under cold running water, or reconstituted with cold phosphate-buffered saline (PBS; pH 7.4) as appropriate. Vaccine to be given with B. pertussis by the subcutaneous route (Group J, Table 1) was recon- stituted with therequired volume (0.25 ml) ofbacterial suspension. Other aqueous preparations were made up to include merozoites and adjuvant in a total volume of I ml per dose, and were given intramuscu- larly. Oil-based adjuvants were emulsified in a Mulsijet syringe,' frequently cooled by plunging into melting ice, and were given intramuscularly. Vaccine was kept on ice and injected promptly after prep- aration. Vaccination regimes and the adjuvants used are shown in Tables 1 and 2. Challenge infections Parasites for challenge infections were bled from acutely infected rhesus monkeys, from an acutely infected splenectomized imported kra monkey, or from a chronically infected United Kingdom-bred kra monkey, and were suitably diluted with PBS to give inocula of 10' or 106 parasites (Tables I and 2). Two monkeys (Group J, Table 1) were challenged directly with parasites retrieved from the deep-freeze. It was known from the dynamics of control infections (11) that these stabilates (Wl, given to Mmu 127; Nuri, given to Mmu 123) contained about 10' and 102 viable parasites, respectively (Mitchell, unpublished data). Challenge was by intravenous inoculation, and infection was detected and monitored by Giemsa- stained thick and thin blood films, as previously described (8). eFrom Mulsijet Inc., Elmburst, IL, USA. E 0 0.o zo 0S I! 0Z 0 z C a 2! a a a 2 lp , I .-a to 4- tC to V- I 2 Is a20 I o 0 0 0 '5 c e c f3Q C a o zE 2Ez° C- 0I 28- xE E EL 22 I c LO co IV to Q 1Z 11- 0 a- .0 a 0 ~ 1 jr 0 0 0 f0 E 4c u JL LLU. X m o 03 r- r- r i I I -a -5 0 0 c, r_l rli 2 C3 2 I 0 m di , e Hh a D F -6 .6 -5 44< < < 4 C: C- .- EL. . L EZEZEiZZ.5ZO E : In S0 E 2 2 2 E2 i u 0 0 2 1* N 1-Z ~ o I-.. V- co C c 0 0 C: C OC 29 S2 is E E- 191 a I 's9 h U t) I z 0 2: o z z i0 -: 3 3 o oo C CO 0 2 Pi F3 D %Z di e - E U 0- az lo~~g n RIX -o n C ..cE a-, 2 cs r_ U 0 z E E cc0 m 'C0 0 to C 3 0 w U' 10 Eco a '9 0 'i m E w a, I 0 ._ 0 : o 0 on w- -o CD 'EI 1 3 I Iv t a QZ a -0 11 a CD -0 &I Co 0 Ql 1s )sE I ui G. Hi. MITCHELL ET AL. Table 2. Vaccination of kra monkeys vvith PIasmodium know/esi merozoltes and results of challenge hrstchalleng.wth 104 Wl parasites Second chalenge with 10' N parasite. Latent Patent Peak pare- Latnt Patent Peok pare- Doys period period aiteruia penod penod sdaemia Monkey Adjuvant veccineteda Day (days) (days) (per 104 RBC) Dayb (days) (day) (per 10' RBC) Ma 67 Saponin 1 &35 65 8 2 C< 1 133 13 5 5. Mz Be Saponin 1 8 35 65 10 2 << 1 133 3 2 <1 MzS C pwvum 1 35 65 24 1 << 1 133 5 chroniC B Mz70 C parvur 1835 66 5 chrontC 150 133 4 chronficc 6 Mz 71 S.pWcsss 1 6&35 65 7 chronicc 60 133 5 chronrcC 40 Mz72 S. petussrs 1 35 65 7 chronicc 46 133 5 chronicC 34 Mz 73 FCA 18E 35 65 14 3 << 1 133 6 chronicc 70 F 34 Non-vaccinated control 4 chroni c 36 3 chronic C 20 a Vaccnated with treeadried merozotes. submequently deeiccated over P,O,. b Alnmonkeys were drug-treated 54 days after first challenga and 40 daysaftersonnd challenge with chloroquine 110 mg/kg) and 4 dady dose of sulfadezine. 15 mg/kg. c Chronically Infected monkeys were patent until radcally cured. RESULTS Pathology at injection sites The gross external pathological findings at the sites of injection in the vaccinated rhesus monkeys are recorded for each animal in Table 1. Where ulceration occurred it was often diffuse and involved both aspects of the limb. Induration of the underlying muscle mass was usually pronounced. In general, lesions were not frankly infected, but those of Mmu 100 and Mmu 108 were found post mortem to be massively infected with Staphylococcus aureus. Of the kra monkeys, only Mz 73, vaccinated using FCA, suffered detectable lesions at its injection sites. The reaction was severe and protracted and both sites were surgically opened to allow drainage on day 51. Local necrosis was most pronounced in this animal. Course of challenge infections in rhesus monkeys The courses of infection in 3 control rhesus mon- keys-Mmu 102, Mmu 103, and 551 -used in these experiments are summarized in Table 3. Monkeys Mmu 102 and Mmu 103 received the same challenge inoculum as the vaccinated monkeys of groupsA and B, and monkey G551 the same challenge inoculum as groups C, D, E, and H (Table 1). All rhesus monkeys except group J were initially challenged With 1O4 Wl parasites. The monkeys of groups A and B were immunized using short-term freeze-dried antigen that had not been secondarily dried, with nor-MDP in FIA, and with FCA, respectively, as adjuvants. None of the monkeys showed a lengthened latent period, all becoming patent simultaneously with the controls on the fourth day of infection. One monkey from each group died, on the thirteenth and fourteenth day of infection, with parasitaemias of 33¾o and 22.5% (Mmu 100 and Mmu 108), whereas control monkeys died on the eighth day with parasitaemias of56% and 62%6. Surviving monkeys of groups A and B suffered maximum parasitaemias between 0.45% and 4.2%o and were patently infected for 15-37 days. Fifty days after initial challenge survivors were rechallenged with parasites of W strain taken from a chronically infected, United Kingdom-born kra monkey. These parasites could not be SICA-typed but were almost certinly not of variant WI. All 4 monkeys survived this heterologous variant second challenge (Table 1). Monkeys of groups C, D, E, and H (Table 1) were immunized using merozoite vaccine that had been stored frozen. Each group received an oil-based adju- vant (groups C and E: groundnut, adjuvant 65; groupsD and H: mineral, FIA) either with added nor- MDP (groups C and D) or M. butyncum (groups E and H). Following challenge, immunized monkeys showed latent periods of 4-7 days (Table 1); the con- trol, G551 (Table 3) had a latent period of4 days. The monkeys ofgroupC (nor-MDP/adjuvant 65) suffered fulminant infections and died with high parasitaemias essentially similar to those of the control infection. Group D monkeys (nor-MDP/FIA) suffered delayed infections (6 days of latency); all 3 died but 2 had low peak parasitaemias (Mmu 114: 6.2%; Mmu 116: 10.2%, Table 1). Monkeys of group E, immunized with M. butyricum in groundnut oil as adjuvant, also showed lengthened latency (5-7 days). One survived, with a peak parasitaemia of 0.81 "fo during 15 days of 192 ADJUVANTS IN EXPERIMENTAL VACCINATION OF AACAQUES Table 3. Plasmodium knowlesi infection of control rhesus monkeys challenged with 104 WI parasites Doy of infecton when. Terminal parasilaamiia Monkey patent died (%) Mrmu 102 4 8 56 Mrnu 103 4 8 & G 51 4 9 % patent infection (Mmu 105, Table 1), while the others suffered fatal infections of 5 and 8 days patency with terminal parasitaemias of 17.4%o and 15.2%o (Mmu 119 and Mlmu 104, Table 1). Group H monkeys received M. butyricum in FIA as adjuvant, i.e., an equivalent of FCA. Infection in these monkeys was delayed (latency 6-7 days), rela- tively mild (peak parasitaemias 0.010io-0.840), and all monkeys survived, with patency lasting 15-19 days. These 3 monkeys and the 1 survivor of group E were challenged 92 days after the initial homologous challenge with 104 parasites of the Nuri strain, taken from an acutely infected rhesus monkey. All 4 animals survived this heterologous strain challenge, only 2 showing patent infection, each for a single day (Mmu 110 and Mmu 117, Table l). Group J monkeys were vaccinated using freeze- dried merozoites, desiccated over P205, with B.pertussis as adjuvant. From results obtained using B.pertussis adjuvant in kra monkeys (see below and Table 2), these rhesus monkeys were not expected to survive challenge and were treated as parasite donors, being infected with stabilate material (see methods) and bled out at terminal parasitaemia. Course of challenge infections in kra mfon keys Challenge infections of the 7 immunized and 1 control kra monkey are summarized in Table 2. Data relating to the first infections of 5 other nor- mal United Kingdom-born kra monkeys (Butcher & Mitchell, unpublished results) are presented in Table 4. Vaccinated kra monkeys received freeze-dried merozoites, desiccated over P205,, together with saponin, C.parvum, B. pertussis, or FCA as adjuvant. Following initial challenge with 104 WI parasites, delayed patency was observed in all the vac- cinated kras, with latent periods 1-20 days longer than in the control. However, both monkeys immunized using B.pertussis (Miz 71 and Mz 72) and one of those immunized using C.parvum (Mz 70) developed chronic low-grade infections (see Table 2) essentially similar to that of the control monkey and those of previously infected normal kra monkeys (Table 4). Parasites weie detectable in blood films from these animals until radical cure 54 days after challenge. Table 4. Plasmodium knowes, infection of United Kingdom-bred kra monkeys challenged with WI parasites Latent Peak period parasiuernia Monkey No of parasites Idays) Patency (%) F 32 10: 5 chronic 0 4 F1B 3 x 10' 6 chronic 2 0 BIRCS 10 4 chronic 3 3 F 29 10' 4 chronic 0 9 F28 5x 104 4 chronic 2 0 Monkeys immunized using saponin (Mz 67 and Mz 68) and FCA (Mz 73), together with one of those receiving C. parvum (Mz 69) suffered only brief and slight patent infection (1-3 days, with fewer than I parasite per 104 erythrocytes). Although free of patent infection, these monkeys were also given cura- tive treatment 54 days after challenge. The control and all vaccinated monkeys were re- challenged 68 days after initial challenge with 10 Nuri strain parasites from an acutely infected, splen- ectomized, imported kra monkey. All developed patent infection after latent periods of 3-13 days, and chronic infection ensued in all except Mz 67 and Mz 68, immunized using saponin adjuvant. These animals suffered low parasitaemias (maxima < 1-5 parasites per 10W erythrocytes) of brief duration (2-5 days patency). Mz 73 (FCA adjuvant) became clini- cally ill during this infection, with a red blood cell count of 2 x 106/mm3 on the fourth day following a peak parasitaemia of 0.70o. Clinical recovery and an improved blood picture occurred spontaneously during the following 10 days. No other animal became anaemic (RBC counts 4-5.5 x 10/mm3) or unwell during the experiments. All were given cura- tive treatment 40 days after the second challenge. DISCUSSION Plasmodiurn knowlesi malaria infection in rhesus and kra macaques The kra or crab-eating macaque, Macacafascicu- (arts, is a natural host of P. knowlesi, suffering a relatively benign and chronic infection (12). The parasite has been isolated from kra populations in the Philippine archipelago (13) and the Malaysian penin- 13 194 G. H. MITCHELL ET AL sula, where it is frequently found (14). For the present experimentalvaccinations ofkra monkeys, individuals bred in the United Kingdom from stocks of Malaysian origin were used, since imported animals might have been exposed previously to P. knowlesi. The initial infection induced in the control kra monkey (F 34, Table 2) was comparable in terms of latent period (4 days) and chronic duration of patency (50 days until cured) with previous first infections of United Kingdom-born kra monkeys (Table 4). The peak parasitaemia, however, was somewhat lower (0.36%) than those recorded in any of the previously infected monkeys (0.40%-3.3%), and was exceeded by the peak parasitaemias recorded from 3 of the vaccinated monkeys (Mz 70, Mz 71, and Mz 72, Table 2). Neither apparent clinical illness nor anaemia is normally associated with infection in the kra monkey. The consistently sbort latent periods of normal animals and the chronicity of ensuing patent infection suggest that an effective immune response to challenge may be indicated by either a delay in the appearance of parasites or the extinction of patent infection. By contrast, the letbality of P. knowlesi malaria in M. mulatta, when induced by blood stage infection, is so nearly uniform that survival is the most significant measure of an effective immune res- ponse. The control rhesus monkeys in the present experiments died with fulminating infections, becom- ing patent on the fourth day of infection and dying 4 or 5 days later (Table 3). However, lengthening of either latency or patency indicates a perturbation of the normal rate of multiplication of parasites, which is effectively logarithmic, and hence shows that the host has mounted a partially effective immune response. Efficacy ofpreserved merozoite vaccine Initially, freslhly prepared merozoite antigen was employed for vaccination (8, 1S). In these early experiments and in subsequent work (16), a total of 13 monkeys were vaccinated using such material, cultured as above (PHA method) or prepared by cell sieving (CS method) (I7), and given on 2 or more occasions with FCA. Of the 5 monkeys initially chal- lenged using parasites homologous with the antigen variant (WI), all survived; 7 of the remaining 8 sur- vived initial heterologous challenge (Table 5). Similar regimes of vaccination have since been employed using modified or stored merozoites (18, 19). The effectiveness of vaccine exposed to 1:1000 formol- saline and freeze-dried without secondary desiccation has been found to be seriously impaired (Table 5) and either formol treatment or incomplete drying alone may also be injurious (Table 5), whereas storage deep- frozen in aqueous medium (but not in oil emulsion; 19) may fully preserve immunogenicity (Table 5). Similarly, limited experience with freeze-dried material, subsequently desiccated over P,O0(Richards & Mitchell, unpublished results), where 3 rhesus monkeys were protected against initial hom- ologous and subsequent heterologous challenges, sug- gests that such treatment may effectively stabilize merozoite vaccine for storage at temperatures of -20°C or higher. Table 5. Vaccination with fresh or modified P. knovwesi merozoites in rhesus monkeys: results of challenge after2 or more injections in Freund's complete adjuvantff Ratio of survivors to number challegd after inmil after indial Treatrment Prepared homologous heterologous of merozoites byb chaNenge chelenge Totals None PHA 4/4 6/6 12/13 (fresh) CS 1/1 1/2 Frozen PHA 1/1 + 3/3f 1/1 5/5 CS - - Formodized d PHA - - 112 C S 0/1 1/1 Formlized and PHA - 216 6i12 freez"rred C S 2/2 2/4 Freeze dried ° PHA 314 + 2/3f 212 715 CS - - a Results from Mitchell et al. (8. J8). Richards et a1 (16) and the present ex- periments b Preparation by differenbal agglutinabon of schizonts (PHA) or cell seving (CS), see text c Stored deep frozen in aqueous suspension. d Exposed to 1:1000 formol-salne ovemight Freeze-dned wihout secondery deseiccation; see text. f Results fromn he present expenments Since freeze-drying without subsequent desiccation over P206 may be an insufficient preservative process, results in the monkeys of groups A and B (Table 1) may reflect the use of an antigen impoverished in this way. The remaining rhesus groups and the kra monkeys must be presumed to have received well-preserved antigen. Nor-MDP, mycobacteria, and oil adjuvants in P. knowlesi vaccination Dissection of the components of mycobacterial cell walls responsible for immuno-adjuvant effects (re- viewed by White, 20) led to the identification (21) and synthesis (22) of the smallest active moiety, N-acetyl- muramyl-L-alanyl-D-isoglutamine (MDP), which, when given with protein antigens in emulsions of water in mineral oil, exhibited an adjuvant potential ADJUVANTS IN EXPERIMENTAL VACCINATION OF MACAQUES comparable to that of whole mycobacteria. In the meantime, many synthetic derivatives of MDP with immunopotentiating properties have become available. Thus, nor-MDP, in which the lactic acid moiety has been replaced by glycolic acid, was shown to display adjuvant properties similar to MDP, but to have a reduced toxicity in nonrodent genera (23 ). The persistence of mineral oil components after injection, and the hazards associated with the extreme irritancy and possible carcinogenicity of such conmpounds, led to the development of an effective but metabolizable and apparently safe adjuvant based on groundnut oil, Adjuvant 65 (reviewed by Hilleman, 24). The present vaccinations of rhesus monkeys (groups A-H, Table 1) were intended to test the ef- ficacy ofnor-MDP as a replacement for mycobacteria in mineral oil, and then the substitution of adjuvant 65 as the oil phase of an adjuvant emulsion. Adjuvant65 alone was known to be an ineffective vehicle for merozoite antigen (19). The initial experiment (groups A and B) suggested that nor-MDP would suc- cessfully replace M. butyricum in Freund's adjuvant, the inconsistency of protection with this combination and with FCA being attributed to poor preservation of merozoite vaccine after incomplete drying. In these groups, the lesions at the injection site may have been aggravated by secondary bacterial infection with S. aureus; alternatively lipases capable of releasing free fatty acid from oil may have been presenL in the merozoite preparation. The inflammatory responses to the release of fatty acid by miciobial enzymes has been a factor restricting the clinical application of FIA to viral vaccines (2). In the second experiment, mcnkeys of group D, re- ceiving nor-MIDP in FIA with deep-frozen stored antigen, were not fully protected, although the course of infection was modified with delay in the develop- ment of patency and its prolongation. MIoreover, in 2 animals terminal parasitaemias were exceptionally low (Mmu 114, 6.2%7o; Mmu 116, 10.27lo). These results confirmed that nor-MDP is part'ally effective in replacing mycobacteria in mineral oil emulsions for P. knowlesi vaccination. Adjuvant 65 combined with Ml. bzaryricumJ (grolp E) protected only 1 of 3 monkeys. This emphasizes the synergism of mycobacterial and mineral oil com- ponents in achieving nearly uniform protection when FCA is used in P. knowlesi immunization. since the highly reactive mycobacteria were musch less effective in the groundnut-oil emulsion. Grouip C monkeys, receiving nor-MDP in adjuvant 65, suffeied unmodi- fied lethal infections, whilst group H animals unho received mycobacteria, from the same source as group E, with FIA (=FCA) suffered mild infection oiil~. Other MDP derivatives and adjuvant vehicles are at present under investigation. The one kra monkey to receive FCA was highly resistant to initial homologous challenge (Table 2) but was clinically ill on subsequent heterologous chal- lenge. This finding is inconsistent with previous limited experience of vaccinating kra monkeys (8). The animal used in this experiment also suffered the worst local reactions to vaccine seen. Bordetella pertussis and Corynebacterium parvum in P. knowlesi immunrzation Neither of these organisms is known to have been used previously as an experimental adjuvant with primate malaria vaccine, but B. perlussis has been effective as an adjuvant with intravenous P. yoezi vaccine in mice (25) and in potentiating the immune response of mice io iriadiated P. berghei (26). The adjuvant effects of both organisms have been reviewed by a WHO Scientific Group (2) and by White (20). Most of them are likely to be enhanced by intravenous administration (27), but the delayed hypersensitivity response to complex particulate anti- gen (sheep erythrocytes) is intensified by subcutaneous administration of B.pertussis adjuvant (28); hence the choice of this route for vaccination of group J rhesus monkeys (Table 1). These animals suffered essentially unmodified infections on challenge. The kra monkeys immunized using C.parvum adjuvant (Mz 69, Mz 70) and B. pertussis adjuvant (Mz 71, Mz 72) all showed some delay in the develop. ment of infection. This may have been due to a par- tially effective inhibitory antibody response, a pos- sibility that is at present under investigation. Chronic infection ensued in all except NIz 69 (Table 3). This monkey must be considered as having mounted a res- ponse largcly effective against homologous challenge, since no such delayed and transient infection has been found in naive kra monkeys (F 34, Table 2; and Table 4). However, in both the monkeys receiving C.par- vum adjuvant, challenge with the distinct Nun strain led to chronic infection, although the peak parasit- aemias were somewhat lower. The relative inefficacy of the coryneforms in this experiment was neverthe- less superior to the action of FCA, since the recipient of this adjuvant (Mz 73) suffered high and chronic parasitaemia with obvious cachexia and anaemia. Saponin as an adjuvant in P. knowlesi vaccination Saponin is a mixture of largely undefined glycoside components, with intense surfactant and irritant paroperties, which has been employed successfully in rodent malaria immuniization (29, 30). The native material must be considered too reactive for human clinical application (although no local lesions were evidenr in tae present experimental monkeys) but a fraction with reduced adverse reactions, Quil A, has 195 196 G. H MITCHELL ET AL. been prepared and tested as an adjuvant in foot-and- mouth disease vaccine (31). In that context, however, elevated antibody response may be the only adjuvant prerequisite, a situation unlikely to pertain with malaria vaccine (19). In the present experiments, unfractionated saponin SPL was very effective as an adjuvant in the kra monkey. Latent periods were lengthened, peak para- sitaemias reduced, and patency shortened in both animals after initial homologous and subsequent heterologous challenges. Effector mechanisms that may be concerned in the immune response to vaccine containing saponin adjuvant are at present under investigation. ACKNOWLEDGEMENTS The authors are most grateful to Dr P. J. Campbell and the staff of the Standards Processing Section, National Institute for Biological Standards and Control, for processing the vaccines, to Dr J. Stanek and Dr A. Hartmann of CIBA-GEIGY, Basic, for synthesizing and kindly providing nor-MDP, and to Dr R. Bomiford of Wellcome Research Laboratories, Beckenham, for kindly providing adjuvants and expert advice on their use. The skilful assistance of Miss M. J. Burnikell, Mrs D. Green, and Mr B. Maples is gratefully acknowledged. This work was supported in part by the Medical Rsearch Council, London. RASUM ESSAI DE DIVERS ADJUVANTS DANS LA VACCINATION EXPPRIMENTALE DE NACAQUES CONTRE LE PALUDISME: NOR-MDP, SAPONINE, CORYWEBACTERIUM PARVtMEET BORDETMLLA PERTUSSIS Des singes rhesus (Macaca mulatta) ont 6tk vaccines au nioyen de m6rozoites de Plasmodium knowlest avec I'adju- vant de Freund incomplet(AFI)- mulsion d'huile minfrale -ou avec l'adjuvant 65- mulsion d'huile d'arachide- auxquels ont Wte ajoutEs respectivement nor-MDP (d&inv6 de muramyl-dipeptide, 6quivalent synth6fique d'un constituant de la membrane mycobact6fienne) et Mycobacterium buty- ricum. Nor-MDP s'est montr& partidellement efficace pour remplacer Its mycobacthries lorsqu'iJ etait ajouti A AFI, mais non lorsqu'il &ait administrt avec 1'adjuvant 65. lees mycobactiries se sont elles-memes r6vel&es peu efficaces avec cet adjuvant; ceci.confirme Ia synergie avec laquelle agissent 1'huile min&rale et lcs mycobact&ies pour induire une protection quasi totale dans les essais d'immunisation par )es m&rozottes de P. knowlesi avec I'adjuvant de Freund complet (AFC). Pour la vaccination des siiiges de l'esp&ce M.fasciculans, on a eu recours A AFC, i la sapomne, A Corynebacteriurn parvuwn et A Bordetella pertussis comme adjuvants. Etant donn6 la nature b6nignc du paludisme A knowlesi chez ces singes, toute comparaison avec l'infection qui frappe les singes rhsus serait malais6e; toutefois, 1'emnploi de la sapo- ninecomme adjuvant a permis, chez M.fascicularis, d'eviter la chronicite de la maladie et d'abaisser les taux maximums de parasit6mie. 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